WO2001009283A2 - Recepteurs chimeres et utilisations associees - Google Patents

Recepteurs chimeres et utilisations associees Download PDF

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WO2001009283A2
WO2001009283A2 PCT/US2000/020714 US0020714W WO0109283A2 WO 2001009283 A2 WO2001009283 A2 WO 2001009283A2 US 0020714 W US0020714 W US 0020714W WO 0109283 A2 WO0109283 A2 WO 0109283A2
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sequence
residue
domain
nucleic acid
plant
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PCT/US2000/020714
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WO2001009283A3 (fr
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Pamela Ronald
Zuhua He
Joanne Chory
Christopher Lamb
Jianming Li
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The Regents Of The University Of California
The Salk Institute For Biological Studies
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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • 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
    • 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
    • C12N15/8281—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 for bacterial resistance
    • 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
    • C12N15/8282—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 for fungal resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates generally to plant molecular biology. In particular, it relates to nucleic acids and methods for conferring disease resistance in plants.
  • Plant pathogens cause hundreds of millions of dollars in damage to crops in the United States annually and cause significantly more damage worldwide.
  • Loci conferring disease resistance have been identified in many plant species. Genetic analysis of many plant-pathogen interactions has demonstrated that plants contain loci that confer resistance against specific races of a pathogen containing a complementary avirulence gene. Molecular characterization of these genes should provide means for conferring disease resistance to a wide variety of crop plants. Bacterial blight disease caused by Xanthomonas spp. infects virtually all crop plants and leads to extensive crop losses worldwide. One source of resistance (Xa21) had been identified and cloned from the wild species Oryza longistaminata (Song et al Science 270:1804-1806 (1995)). The Xa 21 gene is a member of a class of disease resistance genes referred to as "RRK genes".
  • RRK polypeptides which typically comprise an extracellular LRR domain, a transmembrane domain, and a cytoplasmic protein kinase domain (see, WO96/22375 and WO99/09151 for a description of RRK disease resitance genes).
  • the present invention provides nucleic acid molecules comprising a polynucleotide encoding a chimeric RRK receptor, the receptor comprising a heterologous extracellular domain and a kinase domain from a Xa21 polypeptide.
  • the nucleic acid of the invention typically comprise a plant promoter operably linked to the polynucleotide encoding the chimeric RRK receptor.
  • the promoter can be inducible or constitutive.
  • the heterologous extracellular domain comprises an LRR domain, for example from Bril .
  • An exemplary extracellular LRR domain sequence is at least about 70% identical to a sequence from about residue 681 to about residue 3332 in SEQ ID NO: 3.
  • An exemplary kinase domain sequence is at least about 70% identical to a sequence from about residue 2121 to about residue 3918 in SEQ ID NO:l.
  • the heterologous extracellular domain is a hevein domain from RCH10.
  • An exemplary extracellular hevein domain sequence is at least about 70% identical to a sequence from about residue 1928 to about residue 2289 in SEQ ID NO: 5.
  • the kinase domain sequence is usually at least about 70% identical to a sequence from about residue 1870 to about residue 3918 in SEQ ID NO:l.
  • the present invention also provides transgenic plants comprising a recombinant expression cassette comprising a plant promoter operably linked to a polynucleotide sequence encoding a chimeric RRK receptor of the invention.
  • RRK gene is member of a class of plant disease resistance genes which encode RRK polypeptides which typically comprise an extracellular LRR domain, a transmembrane domain, and a cytoplasmic protein kinase domain (as shown in e.g., WO 96/22375).
  • Xa21 polynucleotide sequence is a subsequence or full length polynucleotide sequence of a rice Xa21 gene, which, when present in a transgenic plant confers resistance to Xanthomonas spp. (e.g., X. oryzae) on the plant.
  • Exemplary polynucleotides of the invention include the coding region of the sequences provided below.
  • An Xa21 polynucleotide is typically at least about 3100 nucleotides to about 6500 nucleotides in length, usually from about 4000 to about 4500 nucleotides.
  • a "kinase domain from a Xa21 protein” is a sequence at least substantially identical to a sequence from about residue 708 to about residue 1004 in SEQ ID NO: 2 (see, also, GenBank Accession No. U37133).
  • a "heterologous extracellular domain" in a chimeric receptor of the invention is one that originates from a protein different from the protein from which the kinase domain of the receptor originates.
  • the extracellular domain will comprise an LRR domain or a hevein domain.
  • LRR domain is an extracellular domain which comprises a block of about 23 tandem leucine-rich repeats (LRR) with an average length of 24 amino acids.
  • LRR motif has been implicated in protein-protein interactions and ligand binding in a variety of proteins (see, WO 96/22375).
  • a "Bril polynucleotide sequence” is a subsequence or full length polynucleotide sequence from a Bril gene.
  • a Bril gene encodes brassinosteroid receptor.
  • Brassinosteroids are widely distributed natural products that promote growth and posses plant hormone activity. Brassinosteroids have a number of effects on plants including modulation of response to stress, reproductive processes. There is intensive research being performed to examine the binding proteins, mode of action in regulation of gene expression, biosynthesis, and secondary messengers of these compounds.
  • the encoded protein comprises an extracellular LRR domain containing 25 tandem leucine-rich repeats found in plant disease resistance genes such as RRK genes.
  • RCH10 chitinase polynucleotide sequence refers to a subsequence of full length polynucleotide sequence from a rice RCH10 chitinase gene (Zhu and Lamb Mol Gen. Genet. 226:289-296 (1991).
  • the extracellular domain of this protein comprises a lectin domain homologous to hevein from Hevea brasiliensis.
  • nucleic acid sequence refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. It includes chromosomal DNA, self-replicating plasmids, infectious polymers of DNA or RNA and DNA or RNA that performs a primarily structural role.
  • promoter refers to regions or sequence located upstream and/or downstream from the start of transcription and which are involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.
  • a "plant promoter” is a promoter capable of initiating transcription in plant cells. Such a promoter can be derived from plant genes or from other organisms, such as viruses capable of infecting plant cells.
  • plant includes whole plants, shoot vegetative organs/structures (e.g. leaves, stems and tubers), roots, flowers and floral organs/structures (e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g. vascular tissue, ground tissue, and the like) and cells (e.g. guard cells, egg cells, trichomes and the like), and progeny of same.
  • shoot vegetative organs/structures e.g. leaves, stems and tubers
  • roots e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules
  • seed including embryo, endosperm, and seed coat
  • fruit the mature ovary
  • plant tissue e.g. vascular tissue, ground tissue, and the like
  • cells e.g. guard cells, egg cells, trichomes
  • the class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae. It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid and hemizygous.
  • a polynucleotide sequence is "heterologous to" an organism or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form.
  • a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is not naturally associated with the promoter (e.g. a genetically engineered coding sequence or an allele from a different ecotype or variety).
  • a polynucleotide "exogenous to" an individual plant is a polynucleotide which is introduced into the plant by any means other than by a sexual cross. Examples of means by which this can be accomplished are described below, and include Agrobacterium-mediaied transformation, biolistic methods, electroporation, and the like. Such a plant containing the exogenous nucleic acid is referred to here as a Ti (e.g. in
  • a recombinant expression cassette comprising a promoter operably linked to a second polynucleotide may include a promoter that is heterologous to the second polynucleotide as the result of human manipulation (e.g. , by methods described in Sambrook et al.
  • a recombinant expression cassette may comprise polynucleotides combined in such a way that the polynucleotides are extremely unlikely to be found in nature.
  • human manipulated restriction sites or plasmid vector sequences may flank or separate the promoter from the second polynucleotide.
  • nucleic acid sequences or polypeptides are said to be “identical” if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below.
  • the terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • sequence identity When percentage of sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity.
  • a conservative substitution is given a score between zero and 1.
  • the scoring of conservative substitutions is calculated according to, e.g., the algorithm of Meyers & Miller, Computer Applic. Biol Sci. 4:11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California, USA).
  • substantially identical in the context of two nucleic acids or polypeptides, refers to sequences or subsequences that have at least 60%, preferably 70%, more preferably 80%, most preferably 90-95% nucleotide or amino acid residue identity when aligned for maximum correspondence over a comparison window as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • This definition also refers to the complement of a test sequence, which has substantial sequence or subsequence complementarity when the test sequence has substantial identity to a reference sequence.
  • two polypeptides can also be “substantially identical” if the two polypeptides are immunologically similar. Thus, overall protein structure may be similar while the primary structure of the two polypeptides display significant variation. Therefore a method to measure whether two polypeptides are substantially identical involves measuring the binding of monoclonal or polyclonal antibodies to each polypeptide. Two polypeptides are substantially identical if the antibodies specific for a first polypeptide bind to a second polypeptide with an affinity of at least one third of the affinity for the first polypeptide. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J Mol Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat 7 Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally, Current Protocols in Molecular Biology, F.M. Ausubel et al, eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).
  • HSPs high scoring sequence pairs
  • T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score.
  • Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl Acad. Sci. USA 89:10915 (1989)).
  • 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. Natl 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.
  • a further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, as described below.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions, as described below.
  • Constantly modified variants applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine
  • each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
  • amino acid sequences one of skill will recognize that individual substitutions, in a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
  • sequenceselectively (or specifically) hybridizes to refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).
  • stringent hybridization conditions refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures.
  • the T m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium).
  • Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • a positive signal is at least two times background, preferably 10 time background hybridization.
  • nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
  • genomic DNA or cDNA comprising nucleic acids of the invention can be identified in standard Southern blots under stringent conditions using the nucleic acid sequences disclosed here.
  • suitable stringent conditions for such hybridizations are those which include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37°C, and at least one wash in 0.2X SSC at a temperature of at least about 50°C, usually about 55°C to about 60°C, for 20 minutes, or equivalent conditions.
  • a positive hybridization is at least twice background.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides that they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
  • Exemplary “moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in IX SSC at 45°C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.
  • a further indication that two polynucleotides are substantially identical is if the reference sequence, amplified by a pair of oligonucleotide primers, can then be used as a probe under stringent hybridization conditions to isolate the test sequence from a cDNA or genomic library, or to identify the test sequence in, e.g., an RNA gel or DNA gel blot hybridization analysis.
  • Figure 1 is a schematic representation of chimeric receptors of the invention engineered using the LRR domain of Bril and the kinase domain of Xa21.
  • TM transmembrane region
  • JM juxtamembrane region.
  • Figure 2 is a bar graph showing that brassinosteroid treatment induces cell death in plant cells expressing the chimeric receptors of the invention as compared to controls expressing mutant chimeric receptors and in untransformed cells (TP309).
  • Figure 4 is a bar graph showing that chitin treatment induces cell death in plant cells expressing the chimeric receptors of the invention as compared to controls expressing mutant chimeric receptors and in untransformed cells (TP309).
  • This invention relates to chimeric RRK receptors and genes encoding them.
  • a kinase domain from an Xa21 gene is linked to extracellular domains from heterologous proteins to allow induction of plant disease resistance in response to desired environmental signals.
  • the chimeric receptors of the invention can be used for the discovery of new ligands of the extracellular domains, new functions of the receptor kinases and for engineering new pathogen resistance in plants.
  • oligonucleotide probes based on the sequences disclosed here and in WO 96/22375 and WO 99/09151 can be used to isolate RRK resistance genes.
  • Bril genes can be prepared using the sequences disclosed in WO 98/59039.
  • chitinase genes can be prepared using sequence in Zhu and Lamb Mol Gen. Genet. 226:289-296 (1991).
  • sequences described here and in the prior art are used to identify the desired gene in a cDNA or genomic DNA library.
  • genomic libraries large segments of genomic DNA are generated by random fragmentation, e.g. using restriction endonucleases, and are ligated with vector DNA to form concatemers that can be packaged into the appropriate vector.
  • RNA is isolated from the desired organ, such as leaf and a cDNA library which contains the desired gene transcript is prepared from the mRNA.
  • cDNA may be prepared from mRNA extracted from other tissues in which desired genes or homologs are expressed.
  • the nucleic acids of interest can be amplified from nucleic acid samples using amplification techniques. For instance, polymerase chain reaction (PCR) technology to amplify the sequences of the RRK and related genes directly from genomic DNA, from cDNA, from genomic libraries or cDNA libraries.
  • PCR polymerase chain reaction
  • PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes.
  • PCR Protocols A Guide to Methods and Applications. (Innis, M, Gelfand, D., Sninsky, J. and White, T., eds.), Academic Press, San Diego (1990).
  • Polynucleotides may also be synthesized by well-known techniques as described in the technical literature. See, e.g., Carruthers et al, Cold Spring Harbor Symp. Quant. Biol. 47:411-418 (1982), and Adams et al, J. Am. Chem. Soc. 105:661
  • Double stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence. Isolated sequences prepared as described herein can then be used to provide chimeric receptor gene expression and therefore, for example, enhanced pathogen resistance in desired plants.
  • nucleic acid encoding a functional protein need not have a sequence identical to the exemplified gene or domains disclosed here. Thus, the sequences encoding domains described here need not be full length or identical to those described here, so long as the desired functional domain of the protein is expressed.
  • Modified protein chains can be readily designed utilizing various recombinant DNA techniques well known to those skilled in the art.
  • the chains can vary from the naturally occurring sequence at the primary structure level by amino acid substitutions, additions, deletions, and the like. These modifications can be used in a number of combinations to produce the final modified protein chain.
  • a DNA sequence coding for the desired chimeric receptor polypeptide will be used to construct a recombinant expression cassette which can be introduced into the desired plant.
  • An expression cassette will typically comprise a polynucleotide encoding the chimeric receptor polynucleotide operably linked to transcriptional and translational initiation regulatory sequences which will direct the transcription of the sequence from the polynucleotide in the intended tissues of the transformed plant.
  • a plant promoter fragment may be employed which will direct expression of the chimeric receptor in all tissues of a regenerated plant.
  • Such promoters are referred to herein as "constitutive" promoters and are active under most environmental conditions and states of development or cell differentiation.
  • constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1'- or 2'- promoter derived from T-DNA of Agrobacterium tumafaciens, and other transcription initiation regions from various plant genes known to those of skill.
  • Constitutive promoters and regulatory elements can also be isolated from genes that are expressed constitutively or at least expressed in most if not all tissues of a plant.
  • Such genes include, for example, ACT 11 from Arabidopsis (Huang et al. Plant Mol. Biol. 33:125-139 (1996)), Cat3 from Arabidopsis (GenBank No. U43147, Zhong et al, Mol Gen. Genet. 251 :196-203 (1996)), the gene encoding stearoyl-acyl carrier protein desaturase from Brassica napus (Genbank No. X74782, Solocombe et al. Plant Physiol 104: 1 167-1176 (1994)), GPcl from maize (GenBank No. X15596, Martinez et al. J. Mol. Biol 208:551-565 (1989)), and Gpc2 from maize (GenBank No.
  • the plant promoter may direct expression of a nucleic acid of the invention in a specific tissue, organ or cell type (i.e. tissue-specific promoters) or may be otherwise under more precise environmental or developmental control (i.e. inducible promoters).
  • tissue-specific promoters i.e. tissue-specific promoters
  • inducible promoters examples include pathogen attack, anaerobic conditions, elevated temperature, the presence of light, or application of chemicals/hormones.
  • Tissue-specific promoters can be inducible.
  • tissue-specific promoters may only promote transcription within a certain time frame of developmental stage within that tissue.
  • tissue specific promoters may be active throughout the life cycle of a particular tissue.
  • 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 or cell type, but may also lead to some expression in other tissues as well.
  • promoters under developmental control include promoters that initiate transcription only in certain tissues, such as leaves, roots, fruit, seeds, or flowers.
  • the operation of a promoter may also vary depending on its location in the genome. Thus, an inducible promoter may become fully or partially constitutive in certain locations.
  • promoter sequence elements include the TATA box consensus sequence (e.g. TATAAT), which is usually 20 to 30 base pairs upstream of the transcription start site.
  • TATAAT TATA box consensus sequence
  • promoter element with a series of adenines surrounding the trinucleotide G (orT) N G. J.Messing et al., in Genetic Engineering in Plants, pp.221-227 (Kosage, Meredith and Hollaender, eds. 1983).
  • polyadenylation region at the 3'-end of the chimeric receptor coding region should be included.
  • the polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA.
  • the vector comprising the desired sequences will typically comprise a marker gene which confers a selectable phenotype on plant cells.
  • the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosluforon or Basta.
  • DNA constructs of the invention may be introduced into the genome of the desired plant host by a variety of conventional techniques.
  • the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and micro injection of plant cell protoplasts, or the DNA constructs can be introduced directly to plant tissue using ballistic methods, such as DNA particle bombardment.
  • Microinjection techniques are known in the art and well described in the scientific and patent literature.
  • the introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski et al. EMBO. J. 3:2717-2722 (1984).
  • Electroporation techniques are described in Fromm et al. Proc. Natl. Acad. Sci. USA 82:5824 (1985).
  • Ballistic transformation techniques are described in Klein et al. Nature 327:70-73 (1987).
  • the DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector.
  • 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. See, for example Horsch et al. Science 233:496-498 (1984), and Fraley et al. Proc. Natl. Acad. Sci. USA 80:4803 (1983) and Gene Transfer to Plants, Potrykus, ed. (Springer- Verlag, Berlin 1995).
  • Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype and thus the desired phenotype such as increased seed mass.
  • Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker that has been introduced together with the desired nucleotide sequences.
  • Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee et al. Ann. Rev. of Plant Phys. 38:467-486 (1987).
  • the nucleic acids of the invention can be used to confer desired traits on essentially any plant.
  • the invention has use over a broad range of plants, including species from the genera Anacardium, Arachis, Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Olea, Oryza, Panieum, Pannesetum, Persea, Phaseolus, Pistachia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum, S
  • the expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed. Using known procedures one of skill can screen for plants of the invention by detecting the mRNA or chimeric receptor proteins of the invention in transgenic plants. Means for detecting and quantitating mRNAs or proteins are well known in the art.
  • Plants with enhanced resistance to desired pathogens or other traits can be selected in many ways.
  • One of ordinary skill in the art will recognize that the following methods are but a few of the possibilities.
  • One method of selecting plants with enhanced resistance is to determine resistance of a plant to a specific plant pathogen.
  • Possible pathogens include, but are not limited to, viruses, bacteria, nematodes, fungi or insects (see, e.g., Agrios, Plant Pathology (Academic Press, San Diego, CA) (1988)).
  • pathogens include, but are not limited to, viruses, bacteria, nematodes, fungi or insects (see, e.g., Agrios, Plant Pathology (Academic Press, San Diego, CA) (1988)).
  • resistance responses of plants vary depending on many factors, including what pathogen or plant is used.
  • enhanced resistance is measured by the reduction or elimination of disease symptoms when compared to a control plant.
  • enhanced resistance can also be measured by the production of the hypersensitive response (HR) of the plant (see, e.g., Staskawicz et al. Science 268(5211): 661-7 (1995)). Plants with enhanced resistance can produce an enhanced hypersensitive response relative to control plants.
  • HR hypersensitive response
  • Enhanced resistance can also be determined by measuring the increased expression of a gene operably linked a defense related promoter. Measurement of such expression can be measured by quantitating the accumulation of RNA or subsequent protein product (e.g., using northern or western blot techniques, respectively (see, e.g., Sambrook et al and Ausubel et al).
  • a possible alternate strategy for measuring defense gene promoter expression involves operably linking a reporter gene to the promoter. Reporter gene constructs allow for ease of measurement of expression from the promoter of interest. Examples of reporter genes include: ⁇ -gal, GUS (see, e.g., Jefferson, R. A., et al, EMBO J6: 3901-3907 (1987), green fluorescent protein, luciferase, and others.
  • One of ordinary skill in the art will recognize that the following methods are but a few of the possibilities.
  • One method of selecting plants with enhanced resistance is to determine resistance of a plant to a specific plant stress condition such as heat, cold, or nutritional deprivation.
  • Other possible stress conditions include, but are not limited to, chemicals, metabolic changes (see, e.g., Agrios, Plant Pathology (Academic Press, San Diego, CA) (1988)).
  • resistance responses of plants vary depending on many factors, including what particular stress condition or plant is used.
  • enhanced resistance is measured by the reduction or elimination of stress symptoms when compared to a control plant.
  • Nhel and Kpnl were introduced into Bril residues 681 to 688 and 3327 to 3332 by in vitro mutagenesis.
  • the primer used to introduce the Nhel site is SRG-1, 5'-TCTCTCTCACAGCTAGCTGAGAAATGAAG-3', and the primer for the Kpnl site is SRG-2, 5'-TTCTTCAGGGTACCAATGG-3'.
  • NRG1 a Bril LRR mutant construct called NRGlmL was made by using the Bril mutant binl-3 that has a single amino acid change (G to E) at the residue 2524 (see, Li et al. Cell 90: 929-938 (1997)).
  • NRGlmK Kinase domain mutant construct, NRGlmK, was made by in vitro mutagenesis of the Xa21 kinase domain, changing the amino acid residue K to E in the site 2206 with primer SRG- 13, 5'-GTTGCAGTGGAGGTACTAA-3'.
  • the mutant constructs NRGlmL and NRGlmK were used as controls.
  • Hevein domain from rice chitinase RCH10 sequence 1928 to 2289 see Zhu and Lamb. Mol Gen Gent 226:289-286 (1991)
  • the ⁇ ee Xa21 sequence from 1870 to 3918 were fused to get the chimeric receptor NRG6 (see, Figure 3).
  • the Hevein domain of RCH10 was altered using in vitro mutagenesis with primer SRG-9, 5'-
  • ATCTTATATTTAGCTAGCACACCATGAGA-3' that introduced a Nhel site.
  • a Xmal site was introduced into Xa21 residues 1870-1875 using primer SRG-6, 5'- GGATCTCAATCCCCGGGAATGCCAAACTC-3'.
  • the 0.36 kb Nhel/Xmal fragment containing the Hevein domain o ⁇ RCHIO was combined with the Xmal/Pstl fragment of Xa21, resulting in the chimeric receptor NRG6.
  • a mutant construct NRG9m that consists of the Hevein domain and Xa21 mutant kinase domain with the K to E substitution was made as control.
  • Hevein :Xa21 cell lines were treated with lOOng per ml dissoluble chitin (>5mers) for 24hr. Dye binding increase was measured as above.
  • Rice defense gene (RCH10 and PAL) activation was assayed by Northern blot analysis.
  • RNA samples were prepared from different time points after brassinolide or chitin treatment.
  • a 1.2 kb Nsil/Hindlll fragment of RCH10 (see Zhu and Lamb. Mol Gen Gent 226:289-286 (1991))
  • a 0.4 kb Sphl/Xhol fragment of rice PAL gene ZB8 were used as probes.

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Abstract

L'invention concerne des récepteurs chimères de plantes, utiles pour moduler les réponses de plantes aux pathogènes. Ces récepteurs comprennent un domaine extracellulaire hétérologue ainsi qu'un domaine kinase provenant d'une protéine RRK, telle que Xa21.
PCT/US2000/020714 1999-07-28 2000-07-28 Recepteurs chimeres et utilisations associees WO2001009283A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2503004A (en) * 2012-06-13 2013-12-18 Eberhard Karls Uni Tubingen Chimeric pattern recognition receptors for bacterial plant pathogens such as Xanthomonas
GB2505718A (en) * 2012-09-11 2014-03-12 Eberhard Karls Uni Tubingen Chimeric plant receptors comprising LRR domains from two different receptors
WO2020264237A1 (fr) * 2019-06-27 2020-12-30 Two Blades Foundation Récepteurs de reconnaissance de motif d'atrlp23 modifiés et méthodes d'utilisation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SONG ET AL.: 'A receptor kinase-like protein encoded by the rice disease resistance gene, Xa21' SCIENCE vol. 270, 15 December 1995, pages 1804 - 1806, XP002938371 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2503004A (en) * 2012-06-13 2013-12-18 Eberhard Karls Uni Tubingen Chimeric pattern recognition receptors for bacterial plant pathogens such as Xanthomonas
WO2013186303A1 (fr) * 2012-06-13 2013-12-19 Eberhard Karls Universität Tübingen Récepteur de reconnaissance d'un modèle de plante et ses chimères destinés à être utilisés contre des infections bactériennes
US10676756B2 (en) 2012-06-13 2020-06-09 Eberhard Karls Universitaet Tuebingen Plant pattern recognition receptor and chimeras thereof for use against bacterial infections
GB2505718A (en) * 2012-09-11 2014-03-12 Eberhard Karls Uni Tubingen Chimeric plant receptors comprising LRR domains from two different receptors
GB2505718A9 (en) * 2012-09-11 2014-03-26 Eberhard Karis Uni Tubingen Artificial plant receptors and ligands
WO2020264237A1 (fr) * 2019-06-27 2020-12-30 Two Blades Foundation Récepteurs de reconnaissance de motif d'atrlp23 modifiés et méthodes d'utilisation
CN114450405A (zh) * 2019-06-27 2022-05-06 双刃基金会 工程化atrlp23模式识别受体及使用方法

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