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WO2000008160A2 - Gene de signalisation de la resistance aux maladies des plantes: materiaux et methodes - Google Patents

Gene de signalisation de la resistance aux maladies des plantes: materiaux et methodes Download PDF

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Publication number
WO2000008160A2
WO2000008160A2 PCT/GB1999/002590 GB9902590W WO0008160A2 WO 2000008160 A2 WO2000008160 A2 WO 2000008160A2 GB 9902590 W GB9902590 W GB 9902590W WO 0008160 A2 WO0008160 A2 WO 0008160A2
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Prior art keywords
plant
rarl
polynucleotide
sequence
nucleic acid
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WO2000008160A3 (fr
Inventor
Paul Maria Josef Schulze-Lefert
Ken Shirasu
Thomas Lahaye
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Plant Bioscience Ltd
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Plant Bioscience Ltd
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Priority to AU54288/99A priority Critical patent/AU760571B2/en
Priority to EP99940289A priority patent/EP1102849A2/fr
Priority to JP2000563785A priority patent/JP2002524044A/ja
Priority to CA002337861A priority patent/CA2337861A1/fr
Publication of WO2000008160A2 publication Critical patent/WO2000008160A2/fr
Publication of WO2000008160A3 publication Critical patent/WO2000008160A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • 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

Definitions

  • the present invention relates to a plant disease resistance signalling gene and to materials and methods relating thereto.
  • the invention relates to the Rarl gene of barley and homologues thereof from other species.
  • Plant resistance to pathogens is known to be associated with the induction of a battery of defence-related responses including the production of antimicrobial compounds, the activation of pathogenesis-related (PR) genes, cross-linking of the plant cell wall, and the production of reactive oxygen species (Dixon and Lamb 1990; Hammond-Kosack and Jones 1996) .
  • PR pathogenesis-related
  • HR hypersensitive response
  • R genes isolated from plants fall into two classes, encoding either a variable stretch of leucine rich repeats and a putative nucleotide binding site or encode a leucine rich repeat domain but no nucleotide binding site.
  • a third and fourth class each having only one representative so far, encode proteins with leucine rich repeats and a serine-threonine protein kinase domain, or a protein kinase only.
  • J? genes and the downstream pathways they affect, however, are largely unknown.
  • NDR1 encodes a putative integral membrane protein with unknown biochemical function whereas EDS1 is predicted to encode a putative novel plant lipase .
  • the present inventors have succeeded in cloning the Rarl gene from barley using positional cloning, this despite the fact that map-based isolation of genes from the highly complex barley genome (5.3 x 10 9 bp/haploid genome; (Bennett and Smith 1991) ) poses a major experimental challenge primarily due to unfavourable ratio of genetic and physical distances and due to a high percentage of repetitive noncoding DNA sequences. To date, there has been only one report of a successful map- based isolation of a barley gene (B ⁇ schges et al . 1997) .
  • the invention results from the cloning of the Rarl gene and the provision of homologues and mutant alleles thereof.
  • the invention relates to nucleic acid encoding a polypeptide with Rarl function.
  • Rarl function refers to the ability of the Jarl gene and polypeptide expression products thereof to function in the signalling pathway leading to a plant pathogen defence response and/or cell death and preferably pathogen resistance effected by the direct or indirect interaction of R gene products with pathogen Avr proteins.
  • the term “Rarl function” may be used to refer to sequences which dictate an Rarl phenotype in a plant, the term “Rarl mutant function” or “ rarl function” may be used to refer to forms of Rarl sequences which suppress or cancel an Rarl phenotype in a plant.
  • An rarl mutant phenotype is characterised by the lowering or cancelling of pathogen resistance and/or plant pathogen defence response.
  • Rarl function and rarl function can be determined by assessing the level of defence responses and/or susceptibility of the plant to a pathogen as described above or other suitable alternatives known and available to those skilled in the art.
  • Test plants may be monocotyledenous or dicotyledenous .
  • Suitable monocots include any of barley, rice, wheat, maize or oat, particularly barley.
  • Suitable dicots include Arabidopsis, tobacco, tomato, Brassicas, potato and grape vine .
  • a polynucleotide according to the invention may encode a polypeptide including the amino acid sequence shown in Figure 1.
  • the coding sequence may be that shown included in Figure 1 or it may be a mutant, variant, derivative or allele of the sequence shown.
  • the sequence may differ from that shown by a change which is one or more of addition, insertion, deletion and substitution of one or more nucleotides of the sequence shown. Changes to a nucleotide sequence may result in an amino acid change at the protein level, or not, as determined by the genetic code.
  • nucleic acid according to the present invention may include a sequence different from the sequence shown in Figure 1 yet encode a polypeptide with the same amino acid sequence (i.e. the coding sequence may be "degeneratively equivalent").
  • a polynucleotide according to the invention may include one or more sequences identified as an exon in Figure 4.
  • nucleic acid molecules which include a nucleotide sequence which encodes a polypeptide including an amino acid sequence which although clearly related to a functional Rarl polypeptide (e.g. is immunologically cross reactive with an Rarl polypeptide demonstrating Rarl function, or has characteristic sequence motifs in common with an Rarl polypeptide) no longer has Rarl function.
  • Rarl polypeptide e.g. is immunologically cross reactive with an Rarl polypeptide demonstrating Rarl function, or has characteristic sequence motifs in common with an Rarl polypeptide
  • the present invention provides mutants of Rarl which do not promote a plant pathogen defence response or cell death, and/or pathogen resistance. Plants and plant cells carrying these mutant forms are susceptible to pathogen infection.
  • Rarl mutants, variants, fragments, derivatives, alleles and homologues of types which raise resistance and of types which lower resistance may both be of practical value depending on the situation.
  • the major interest will be one of raising plant resistance to pathogens .
  • homologues of the particular Rarl sequences provided herein are provided by the present invention as are mutants, variants, fragments and derivatives of such homologues (and comments made above in relation to such mutants etc also apply in relation to mutants etc of homologues) .
  • Such homologues are readily obtainable by use of the disclosures made herein.
  • the present invention also extends to nucleic acid molecules which include a nucleic acid sequence encoding an Rarl homologue obtainable using a nucleotide sequence derived from, or as shown in Figure 1 or Figure 4, or obtainable using amino acid sequence shown in Figure 1 or Figure 4.
  • the Rarl homologue may at the nucleotide level have homology with a nucleotide sequence of Figure 1, or may encode a polypeptide which has homology with the polypeptide of which the amino acid sequence is shown in Figure 1, preferably at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80% homology, or at least about 90% homology. Most preferably at least about 95% or greater homology. (Determination of homology at the amino acid level is discussed further below. )
  • a polypeptide allele, variant, derivative, mutant derivative, mutant or homologue of the specific sequence may show little overall homology, say about 20%, or about 25%, or about 30%, or about 35%, or about 40% or about 45%, with the specific amino acid sequence of Figure 1.
  • the amino acid homology may be much higher.
  • Putative functionally significant domains or regions can be identified using processes of bioinformatics, including comparison of the sequences of homologues.
  • Functionally significant domains or regions of different polypeptides may be combined for expression from encoding nucleic acid as a fusion protein.
  • particularly advantageous or desirable properties of different homologues may be combined in a hybrid protein, such that the resultant expression product, with Rarl or Rarl function, may include fragments of various parent proteins.
  • Each nucleotide sequence of Figure 7 represents a further aspect of the present invention, and polynucleotides comprising a sequence as shown may be employed in various aspects and embodiments disclosed herein.
  • Rarl-derived oligonucleotide primers may be used to isolate Rarl homologues from many different plants, including monocots and dictors, such as barley, wheat, maize, oats, rice, tomatoes, melons, cucurbi taceae, Brassicaceae, capsicums, lettuces, grape vines, ornamentals.
  • a corresponding gene may be expressed as an antisense construct to assess its importance in resistance to agronomically important diseases such as Puccinia hordei (leaf rust) , Rhynchosproium secalis (scald) , Pyrenophera teres (net blotch) , Heterodera avenae (barley cereal cyst namatode) , Drechslera teres, Powdery mildew and yellow dwarf virus (e.g. barley yellow dwarf virus) .
  • Puccinia hordei leaf rust
  • Rhynchosproium secalis scald
  • Pyrenophera teres net blotch
  • Heterodera avenae barley cereal cyst namatode
  • Drechslera teres Powdery mildew and yellow dwarf virus (e.g. barley yellow dwarf virus) .
  • nucleotide sequence information provided herein, or any part thereof, may be used in a data-base search to find homologous sequences, expression products of which can be tested for Rarl (or rarl) function. These may have ability to complement an Rarl (or rarl) phenotype in a plant or may, upon expression in a plant, confer such a phenotype.
  • Rarl or rarl
  • the .Rarl cDNA or part of it may be used as a bait in an interaction trap assay, such as the yeast two-hybrid system, to isolate other disease resistance signalling components that are hitherto unknown. These present further targets for pathway manipulation towards improved disease resistance.
  • homologues may be exploited in the identification of further homologues, for example using oligonucleotides (e.g. a degenerate pool) designed on the basis of sequence conservation or PCR primers .
  • oligonucleotides e.g. a degenerate pool
  • Primers useful in aspects of the present invention include "AtRarl 5'" and “AtRarl 3'", the sequences of which are given below.
  • the present invention provides a method of identifying or a method of cloning an Rarl homologue, e.g. from a species other than Barley, the method employing a nucleotide sequence derived from that shown in Figure 1 or Figure 4.
  • a method may include providing a preparation of plant cell nucleic acid, providing a nucleic acid molecule having a nucleotide sequence substantially as shown herein or complementary to a nucleotide sequence substantially as shown herein, preferably from within the coding sequence (e.g.
  • Target or candidate nucleic acid may, for example, include genomic DNA, cDNA or RNA (or a mixture of any of these preferably as a library) obtainable from an organism known to contain or suspected of containing such nucleic acid, either monocotyledonous or dicotyledonous .
  • genomic DNA e.g., genomic DNA, cDNA or RNA (or a mixture of any of these preferably as a library) obtainable from an organism known to contain or suspected of containing such nucleic acid, either monocotyledonous or dicotyledonous .
  • the complexity of a nucleic acid library may be reduced by creating a cDNA library for example using RT- PCR or by using the phenol emulsion reassociation technique (Clarke et al . (1992) NAR 20, 1289-1292) on a genomic library.
  • Successful hybridisation may be identified and target/candidate nucleic acid isolated for further investigation and/or use.
  • Hybridisation of nucleic acid molecule to a Rarl gene or homologue may be determined or identified indirectly, e.g. using a nucleic acid amplification reaction, particularly the polymerase chain reaction (PCR) .
  • PCR requires the use of two primers to specifically amplify target nucleic acid, so preferably two nucleic acid molecules with sequences characteristic of Rarl are employed. However, if RACE is used only one such primer may be needed.
  • Hybridisation may be also be determined (optionally in conjunction with an amplification technique such as PCR) by probing with nucleic acid and identifying positive hybridisation under suitably stringent conditions (in accordance with known techniques) .
  • preferred conditions are those which are stringent enough for there to be a simple pattern with a small number of hybridisations identified as positive which can be investigated further. It is well known in the art to increase stringency of hybridisation gradually until only a few positive clones remain.
  • Binding of a probe to target nucleic acid may be measured using any of a variety of techniques at the disposal of those skilled in the art.
  • probes may be radioactively, fluorescently or enzymatically labelled.
  • Other methods not employing labelling of probe include examination of restriction fragment length polymorphisms, amplification using PCR, RNAase cleavage and allele specific oligonucleotide probing.
  • Probing may employ the standard Southern blotting technique. For instance DNA may be extracted from cells and digested with different restriction enzymes. Restriction fragments may then be separated by electrophoresis on an agarose gel, before denaturation and transfer to a nitrocellulose filter. Labelled probe may be hybridised to the DNA fragments on the filter and binding determined. DNA for probing may be prepared from RNA preparations from cells by techniques such as reverse-transcriptase-PCR.
  • Preliminary experiments may be performed by hybridising under low stringency conditions various probes to Southern blots of DNA digested with restriction enzymes.
  • preferred conditions are those which are stringent enough for there to be a simple pattern with a small number of hybridisations identified as positive which can be investigated further. It is well known in the art to increase stringency of hybridisation gradually until only a few positive clones remain. Suitable conditions would be achieved when a large number of hybridising fragments were obtained while the background hybridisation was low. Using these conditions nucleic acid libraries, e.g. cDNA libraries representative of expressed sequences, may be searched.
  • the screening is carried out at about 37°C, a formamide concentration of about 20%, and a salt concentration of about 5 X SSC, or a temperature of about 50°C and a salt concentration of about 2 X SSPE.
  • Suitable conditions include, e.g. for detection of sequences that are about 80-90% identical, hybridization overnight at 42°C in 0.25M Na 2 HP0 4 , pH 7.2 , 6.5% SDS, 10% dextran sulfate and a final wash at 55°C in 0. IX SSC, 0.1% SDS.
  • suitable conditions include hybridization overnight at 65°C in 0.25M Na 2 HP0 4 , pH 7.2 , 6.5% SDS, 10% dextran sulfate and a final wash at 60°C in 0. IX SSC, 0.1% SDS.
  • filters are washed as follows: (1) 5 minutes at room temperature in 2X SSC and 1% SDS; (2) 15 minutes at room temperature in 2X SSC and 0.1% SDS; (3) 30 minutes - 1 hour at 37°C in IX SSC and 1% SDS; (4) 2 hours at 42-65°C in IX SSC and 1% SDS, changing the solution every 30 minutes.
  • T m 81.5°C + 16.6Log [Na+] + 0.41 (% G+C) - 0.63 (% formamide) - 600/#bp in duplex.
  • the T m is 57°C.
  • the T m of a DNA duplex decreases by 1 - 1.5°C with every 1% decrease in homology.
  • targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42°C.
  • Such a sequence would be considered substantially homologous to the nucleic acid sequence of the present invention.
  • suitable conditions include, e.g. for detection of sequences that are about 80-90% identical, hybridization overnight at 42 °C in 0.25M Na 2 HP0 4 , pH 7.2 , 6.5% SDS, 10% dextran sulfate and a final wash at 55°C in 0. IX SSC, 0.1% SDS.
  • suitable conditions include hybridization overnight at 65°C in 0.25M Na 2 HP0 4 , pH 7.2 , 6.5% SDS, 10% dextran sulfate and a final wash at 60°C in 0. IX SSC, 0.1% SDS.
  • An alternative, which may be particularly appropriate with plant nucleic acid preparations, is a solution of 5x SSPE (final 0.9 M NaCI , 0.05M sodium phosphate, 0.005M EDTA pH 7.7), 5X Denhardt's solution, 0.5% SDS, at 65°C overnight, (for high stringency, highly similar sequences) or 50°C (for low stringency, less similar sequences). Washes in 0.2x SSC/0.1% SDS at 65°C for high stringency, alternatively at 50-60°C in lx SSC/0.1% SDS for low stringency.
  • the present invention extends to nucleic acid selectively hybridisable under high stringency with nucleic acid identified herein, e.g. the coding sequence of Figure 1, the sequence of Figure 4 or the sequence of Figure 5C or Figure 5D.
  • PCR techniques for the amplification of nucleic acid are described in US Patent No. 4,683,195 and Saiki et al . Science 239: 487-491 (1988). PCR includes steps of denaturation of template nucleic acid (if double-stranded) , annealing of primer to target, and polymerisation.
  • the nucleic acid probed or used as template in the amplification reaction may be genomic DNA, cDNA or RNA.
  • PCR may be used to amplify specific sequences from genomic DNA, specific RNA sequences and cDNA transcribed from mRNA. References for the general use of PCR techniques include Mullis et al , Cold Spring
  • a PCR band may contain a complex mix of products. Individual products may be cloned and each screened for linkage to such known genes that are segregating in progeny that showed a polymorphism for this probe. Alternatively, the PCR product may be treated in a way that enables one to display the polymorphism on a denaturing polyacrylamide DNA sequencing gel with specific bands that are linked to the gene being preselected prior to cloning.
  • a candidate PCR band may be used to isolate clones which may be inspected for other features and homologies to Rarl/Rarl or other related gene. It may subsequently be analysed by transformation to assess its function on introduction into a disease sensitive variety of the plant of interest. Alternatively, the PCR band or sequences derived by analysing it may be used to assist plant breeders in monitoring the segregation of a useful resistance gene. These techniques are of general applicability to the identification of genes able to alter a plant's resistance to a pathogen.
  • Preferred amino acid sequences suitable for use in the design of probes or PCR primers are sequences conserved (completely, substantially or partly) between at least two Rarl peptides or polypeptides encoded by genes involved in the signalling of a defence response in a plant . conserveed sequences may be identified using information contained herein, for instance in Figure 3.
  • oligonucleotide probes or primers may be designed (when working from amino acid sequence information, taking into account the degeneracy of the genetic code and where appropriate, codon usage of the organism) .
  • a gene or fragment thereof identified as being that to which a said nucleic acid molecule hybridises may be isolated and/or purified and may be subsequently investigated for ability to alter a plant's resistance to a pathogen. If the identified nucleic acid is a fragment of a gene, the fragment may be used (e.g. by probing and/or PCR) in subsequent cloning of the full- length gene, which may be a full-length coding sequence. Inserts may be prepared from partial cDNA clones and used to screen cDNA libraries. The full-length clones isolated may be subcloned into expression vectors and activity assayed by introduction into suitable host cells and/or sequenced. It may be necessary for one or more gene fragments to be ligated to generate a full-length coding sequence.
  • Molecules found to manipulate genes with ability to alter a plant's resistance to infection may be used as such, i.e. to alter a plant's resistance to a pathogen.
  • Nucleic acid obtained and obtainable using a method as disclosed herein is provided in various aspects of the present invention.
  • the present application also provides oligonucleotides based on either an Rarl nucleotide sequence as provided herein or an Rarl nucleotide sequence obtainable in accordance with the disclosures and suggestions herein.
  • the oligonucleotides may be of a length suitable for use as primers in an amplification reaction, or they may be suitable for use as hybridization fishing probes.
  • an oligonucleotide in accordance with the invention e.g. for use in nucleic acid amplification, has about 10 or fewer codons (e.g. 6, 7 or 8), i.e. is about 30 or fewer nucleotides in length (e.g. 18, 21 or 24) .
  • a probe or primer may be about 20-30 nucleotides in length.
  • Nucleic acid molecules and vectors according to the present invention may be provided in a form isolated and/or purified from their natural environment, in substantially pure or homogeneous, or free or substantially free of nucleic acid and or genes of the species of interest or origin other than the relevant sequence.
  • Nucleic acid according to the present invention may include cDNA, RNA, genomic DNA and may be wholly or partially synthetic. The term "isolate" where used may encompass any of these possibilities.
  • Nucleic acid as herein provided or obtainable by use of the disclosures herein may be the subject of alteration by way of one or more of addition, insertion, deletion or substitution of nucleotides with or without altering the encoded amino acid sequence (by virtue of the degeneracy of the genetic code) .
  • Such altered forms of Rarl nucleotide sequences as herein provided or obtainable by use of the disclosures herein can be easily and routinely tested for both Rarl function and Rarl function in accordance with standard techniques which basically examine plants or plant cells carrying the mutant, derivative or variant for a altered defence response to an appropriate pathogen.
  • the nucleic acid molecule may be in the form of a recombinant and preferably replicable vector for example a plasmid, cosmid, phage or binary vector, e.g. suitable for use with Agrobacterium.
  • the nucleic acid may be under the control of an appropriate promoter and regulatory elements for expression in a host cell such as a microbial, e.g. bacterial, or plant cell. In the case of genomic DNA, this may contain its own promoter and regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter and regulatory elements for expression in the host cell.
  • the nucleotide sequence of Figure 1 (for example) may be placed under the control of a promoter other than that of the Barley Rarl gene.
  • a Rarl homologue sequence from another species may be operably linked to a promoter other than that with which it is naturally associated.
  • a vector including nucleic acid according to the present invention need not include a promoter, particularly if the vector is to be used to introduce the nucleic acid into cells for recombination into the genome .
  • the nucleic acid as provided by the present invention may be placed under the control of an inducible gene promoter thus placing expression under the control of the user.
  • the present invention provides a gene construct including an inducible promoter operatively linked to a nucleotide sequence provided by the present invention. As discussed, this enables control of expression of the gene.
  • the invention also provides plants transformed with said gene construct and methods including introduction of such a construct into a plant cell and/or induction of expression of a construct within a plant cell, e.g by application of a suitable stimulus, such as an effective exogenous inducer.
  • inducible as applied to a promoter is well understood by those skilled in the art. In essence, expression under the control of an inducible promoter is "switched on” or increased in response to an applied stimulus (which may be generated within a cell or provided exogenously) . The nature of the stimulus varies between promoters. Some inducible promoters cause little or undetectable levels of expression (or no expression) in the absence of the appropriate stimulus. Other inducible promoters cause detectable constitutive expression in the absence of the stimulus. Whatever the level of expression is in the absence of the stimulus, expression from any inducible promoter is increased in the presence of the correct stimulus.
  • an inducible (or “switchable” ) promoter may be used which causes a basic level of expression in the absence of the stimulus which level is too low to bring about a desired phenotype (and may in fact be zero) .
  • expression is increased (or switched on) to a level which brings about the desired phenotype.
  • an inducible promoter is the ethanol inducible gene switch disclosed in Caddick et al (1998) Nature Biotechnology 16: 177-180. Many other examples will be known to those skilled in the art.
  • Suitable promoters may include the Cauliflower Mosaic Virus 35S (CaMV 35S) gene promoter that is expressed at a high level in virtually all plant tissues (Benfey et al ,
  • the cauliflower meri 5 promoter that is expressed in the vegetative apical meristem as well as several well localised positions in the plant body, e.g. inner phloem, flower primordia, branching points in root and shoot (Medford, J.I. (1992) Plant Cell 4, 1029-1039; Medford et al , (1991) Plant Cell 3, 359-370) and the Arabidopsis thaliana LEAFY promoter that is expressed very early in flower development (Weigel et al , (1992) Cell 69, 843-859) .
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • appropriate regulatory sequences including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • Molecular Cloning a Laboratory Manual : 2nd edition, Sambrook et al , 1989, Cold Spring Harbor Laboratory Press.
  • Many known techniques and protocols for manipulation of nucleic acid for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Second Edition, Ausubel et al .
  • Selectable genetic markers may be used consisting of chimaeric genes that confer selectable phenotypes such as resistance to antibiotics such as kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate .
  • nucleic acid to be inserted should be assembled within a construct which contains effective regulatory elements which will drive transcription. There must be available a method of transporting the construct into the cell. Once the construct is within the cell membrane, integration into the endogenous chromosomal material either will or will not occur. Finally, as far as plants are concerned the target cell type must be such that cells can be regenerated into whole plants.
  • Plants transformed with the DNA segment containing the sequence may be produced by standard techniques which are already known for the genetic manipulation of plants.
  • DNA can be transformed into plant cells using any suitable technology, such as a disarmed Ti-plasmid vector carried by Agrobacterium exploiting its natural gene transfer ability (EP-A-270355, EP-A-0116718 , NAR 12(22) 8711 -87215 1984), particle or microprojectile bombardment (US 5100792, EP-A- 444882, EP-A-434616) microinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green et al .
  • a disarmed Ti-plasmid vector carried by Agrobacterium exploiting its natural gene transfer ability (EP-A-270355, EP-A-0116718 , NAR 12(22) 8711 -87215 1984), particle or microprojectile bombardment (US 5100792, EP-A- 444882, EP-A-434616) microinjection (WO
  • Agrobacterium transformation is widely used by those skilled in the art to transform dicotyledonous species. Production of stable, fertile transgenic plants in almost all economically relevant monocot plants is also now routine: (Toriyama, et al . (1988) Bio/Technology 6, 1072-1074; Zhang, et al . (1988) Plant Cell Rep . 7, 379-384; Zhang, et al . (1988) Theor Appl Genet 76, 835-840; Shimamoto, et al . (1989) Nature 338, 274-276; Datta, et al . (1990) Bio/Technology 8, 736-740; Christou, et al .
  • Microprojectile bombardment, electroporation and direct DNA uptake are preferred where Agrobacterium is inefficient or ineffective.
  • a combination of different techniques may be employed to enhance the efficiency of the transformation process, e.g. bombardment with Agrobacterium coated microparticles (EP-A-486234) or microprojectile bombardment to induce wounding followed by co-cultivation with Agrobacterium (EP-A-486233) .
  • a plant may be regenerated, e.g. from single cells, callus tissue or leaf discs, as is standard in the art. Almost any plant can be entirely regenerated from cells, tissues and organs of the plant. Available techniques are reviewed in Vasil et al . , Cell Cul ture and Somatic Cell Genetics of Plants, Vol I, II and XXX, Laboratory Procedures and Their Applications, Academic Press, 1984, and Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989.
  • the invention further encompasses a host cell transformed with a vector as set forth above, especially a plant or a microbial cell.
  • a host cell such as a plant cell, including a nucleotide sequence as herein indicated is provided.
  • the nucleotide sequence may be incorporated within the chromosome.
  • a plant cell having incorporated into its genome a nucleotide sequence, particularly a heterologous nucleotide sequence, as provided by the present invention under operative control of a regulatory sequence for control of expression.
  • the coding sequence may be operably linked to one or more regulatory sequences which may be heterologous or foreign to the gene, such as not naturally associated with the gene for its expression.
  • the nucleotide sequence according to the invention may be placed under the control of an externally inducible gene promoter to place expression under the control of the user.
  • a further aspect of the present invention provides a method of making such a plant cell involving introduction of nucleotide sequence or a suitable vector including the sequence of nucleotides into a plant cell and causing or allowing recombination between the vector and the plant cell genome to introduce the sequence of nucleotides into the genome.
  • the invention extends to plant cells containing a nucleotide sequence according to the invention as a result of introduction of the nucleotide sequence into an ancestor cell.
  • heterologous may be used to indicate that the gene/sequence of nucleotides in question have been introduced into said cells of the plant or an ancestor thereof, using genetic engineering, ie by human intervention.
  • a transgenic plant cell i.e. transgenic for the nucleotide sequence in question, may be provided.
  • the transgene may be on an extra- genomic vector or incorporated, preferably stably, into the genome.
  • a heterologous gene may replace an endogenous equivalent gene, ie one which normally performs the same or a similar function, or the inserted sequence may be additional to the endogenous gene or other sequence .
  • nucleotide sequences heterologous, or exogenous or foreign, to a plant cell may be non-naturally occurring in cells of that type, variety or species.
  • a nucleotide sequence may include a coding sequence of or derived from a particular type of plant cell or species or variety of plant, placed within the context of a plant cell of a different type or species or variety of plant.
  • nucleotide sequence to be placed within a cell in which it or a homologue is found naturally, but wherein the nucleotide sequence is linked and/or adjacent to nucleic acid which does not occur naturally within the cell, or cells of that type or species or variety of plant, such as operably linked to one or more regulatory sequences, such as a promoter sequence, for control of expression.
  • a sequence within a plant or other host cell may be identifiably heterologous, exogenous or foreign.
  • Plants which include a plant cell according to the invention are also provided, along with any part or propagule thereof, seed, selfed or hybrid progeny and descendants.
  • transgenic crop plants which have been engineered to carry genes identified as stated above.
  • suitable plants include tobacco, cucurbits, carrot, vegetable brassica, melons, capsicums, grape vines, lettuce, strawberry, oilseed brassica, sugar beet, wheat, barley, maize, rice, soyabeans, peas, sorghum, sunflower, tomato, potato, pepper, chrysanthemum, carnation, poplar, eucalyptus and pine.
  • a plant according to the present invention may be one which does not breed true in one or more properties. Plant varieties may be excluded, particularly registrable plant varieties according to Plant Breeders' Rights. It is noted that a plant need not be considered a "plant variety” simply because it contains stably within its genome a transgene, introduced into a cell of the plant or an ancestor thereof.
  • the present invention provides any clone of such a plant, seed, selfed or hybrid progeny and descendants, and any part of any of these, such as cuttings, seed.
  • the invention provides any plant propagule, that is any part which may be used in reproduction or propagation, sexual or asexual, including cuttings, seed and so on.
  • the present invention also encompasses the polypeptide expression product of a nucleic acid molecule according to the invention as disclosed herein or obtainable in accordance with the information and suggestions herein. Also provided are methods of making such an expression product by expression from a nucleotide sequence encoding therefore under suitable conditions in suitable host cells e.g. E. coli . Those skilled in the art are well able to construct vectors and design protocols and systems for expression and recovery of products of recombinant gene expression.
  • a preferred polypeptide includes the amino acid sequence shown in Figure 1.
  • a polypeptide according to the present invention may be an allele, variant, fragment, derivative, mutant or homologue of a polypeptide as shown in Figure 1.
  • the allele, variant, fragment, derivative, mutant or homologue may have substantially the Rarl function of the amino acid sequence shown in Figure 1 or may be a rarl mutant .
  • polypeptides which although clearly related to a functional Rarl polypeptide (e.g. they are immunologically cross reactive with an Rarl polypeptide demonstrating Rarl function, or they have characteristic sequence motifs in common with an Rarl polypeptide) no longer have Rarl function.
  • the present invention provides variant forms of Rarl polypeptides, such as those resulting from the rarl -1 and rarl -2 mutations identified herein. Plants and plant cells carrying these mutant forms are susceptible to pathogen ingress.
  • “Homology” in relation to an amino acid sequence may be used to refer to identity or similarity, preferably identity. As noted already above, high level of amino acid identity may be limited to functionally significant domains or regions, e.g. any of the domains identified herein (e.g. see Figure 6) .
  • homologues of the particular Rarl polypeptide sequences provided herein are provided by the present invention, as are mutants, variants, fragments and derivatives of such homologues .
  • Such homologues are readily obtainable by use of the disclosures made herein.
  • the present invention also extends to polypetides which include an amino acid sequence with Rarl function obtainable using sequence information as provided herein.
  • the Rarl homologue may at the amino acid level have homology with the amino acid sequence of Figure 1, preferably at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80% homology, or at least about 85 %, or at least about 88% homology, or at least about 90% homology. Most preferably at least about 95% or greater homology.
  • an allele, variant, derivative, mutant derivative, mutant or homologue of the specific sequence may show little overall homology, say about 20%, or about 25%, or about 30%, or about 35%, or about 40% or about 45%, with the specific sequence.
  • the amino acid homology may be much higher.
  • Putative functionally significant domains or regions can be identified using processes of bioinformatics, including comparison of the sequences of homologues.
  • Functionally significant domains or regions of different polypeptides may be combined for expression from encoding nucleic acid as a fusion protein.
  • particularly advantageous or desirable properties of different homologues may be combined in a hybrid protein, such that the resultant expression product, with Rarl or Rarl function, may include fragments of various parent proteins.
  • Individual domains and fragments of Rarl polypeptide are shown in Figure 6 and these, also derivatives, variants and homologues as noted, are useful in various aspects and embodiments of the invention, for instance in the activation of cell death and/or downstream resistance responses.
  • Similarity of amino acid sequences may be as defined and determined by the TBLASTN program, of Altschul et al . (1990) J " . Mol . Biol . 215: 403-10, which is in standard use in the art.
  • TBLASTN 2.0 may be used with Matrix BL0SUM62 and GAP penalties: existence: 11, extension: 1.
  • Another standard program that may be used is BestFit, which is part of the Wisconsin Package, Version 8, September 1994, (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA, Wisconsin 53711) . BestFit makes an optimal alignment of the best segment of similarity between two sequences. Optimal alignments are found by inserting gaps to maximize the number of matches using the local homology algorithm of Smith and Waterman (Adv.
  • GAP Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps.
  • GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps.
  • a gap creation penalty of 3 and gap extension penalty of 0.1 may be used.
  • FASTA which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448) is a further alternative.
  • Rarl polypeptides and mutants, variants, fragments, derivatives, alleles and homologues thereof e.g. produced recombinantly by expression from encoding nucleic acid therefor, may be used to raise antibodies employing techniques which are standard in the art .
  • Antibodies and polypeptides including antigen-binding fragments of antibodies may be used in identifying homologues of the sequences specifically provided herein as discussed further below.
  • Methods of producing antibodies include immunising a mammal (e.g. human, mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or a fragment thereof.
  • Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and might be screened, preferably using binding of antibody to antigen of interest. For instance, Western blotting techniques or immunoprecipitation may be used (Armitage et al , 1992, Nature 357: 80-82). Antibodies may be polyclonal or monoclonal.
  • antibodies with appropriate binding specificity may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance see WO92/01047.
  • Antibodies raised to a polypeptide or peptide can be used in the identification and/or isolation of homologous polypeptides, and then the encoding genes.
  • the present invention provides a method of identifying or isolating a polypeptide with Rarl function or Rarl function (in accordance with embodiments disclosed herein) , including screening candidate peptides or polypeptides with a polypeptide including the antigen-binding domain of an antibody (for example whole antibody or a fragment thereof) which is able to bind an Rarl or Rarl peptide, polypeptide or fragment, variant or variant thereof or preferably has binding specificity for such a peptide or polypeptide, such as having an amino acid sequence identified herein.
  • an antibody for example whole antibody or a fragment thereof
  • Specific binding members such as antibodies and polypeptides including antigen binding domains of antibodies that bind and are preferably specific for a Rarl or Rarl peptide or polypeptide or mutant, variant or derivative thereof represent further aspects of the present invention, as do their use and methods which employ them.
  • Candidate peptides or polypeptides for screening may for instance be the products of an expression library created using nucleic acid derived from an plant of interest, or may be the product of a purification process from a natural source.
  • a peptide or polypeptide found to bind the antibody may be isolated and then may be subject to amino acid sequencing. Any suitable technique may be used to sequence the peptide or polypeptide either wholly or partially (for instance a fragment of a polypeptide may be sequenced) .
  • Amino acid sequence information may be used in obtaining nucleic acid encoding the peptide or polypeptide, for instance by designing one or more oligonucleotides (e.g. a degenerate pool of oligonucleotides) for use as probes or primers in hybridisation to candidate nucleic acid, or by searching computer sequence databases, as discussed further below.
  • the invention further provides a method of promoting cell death and/or a plant pathogen defence response in a plant which includes expressing a heterologous nucleic acid sequence with Rarl function as discussed, within cells of the plant .
  • the invention further provides a method of raising pathogen resistance in a plant which includes expressing a heterologous nucleic acid sequence with Rarl function as discussed, within cells of the plant.
  • Such methods may be achieved by expression from a nucleotide sequence encoding an amino acid sequence conferring an Rarl function within cells of a plant (thereby producing the encoded polypeptide) , following an earlier step of introduction of the nucleotide sequence into a cell of the plant or an ancestor thereof. Such a method may raise the plant's resistance to pathogen.
  • Manipulation of expression of the Rarl transcript or Rarl protein may be used to enhance resistance to a broad spectrum of pathogens in different plants. This may be achieved by over expression using a highly active plant promoter such as the CaMV-35S promoter. Alternatively, Rarl may be attached to a pathogen-inducible promoter (see discussion below) , allowing greater expression in challenged cells. Increased disease resistance may occur in the absence of a hypersensitive response (HR) that may have possible deleterious effects to the plant in terms of general vigour and yield.
  • HR hypersensitive response
  • a gene stably incorporated into the genome of a plant is passed from generation to generation to descendants of the plant, cells of which descendants may express the encoded polypeptide and so may have enhanced pathogen resistance or pathogen susceptibility.
  • Pathogen resistance may be determined by assessing compatibility of a pathogen as earlier mentioned.
  • the invention further provides a method which includes expression from a nucleic acid encoding the amino acid sequence of Figure 1 or a mutant, allele or derivative of the sequence (which may have Rarl function) within cells of a plant (thereby producing the encoded polypeptide) , following an earlier step of introduction of the nucleic acid into a cell of the plant or an ancestor thereof.
  • a method may raise the plant's resistance to one or more pathogens.
  • the method may be used in combination with an avr gene according to any of the methods described in W091/15585 (Mogen) or, more preferably, PCT/GB95/01075 (published as WO 95/31564) , or any other gene involved in conferring pathogen resistance.
  • alteration of resistance may be achieved by introduction of the nucleotide sequence in a sense orientation.
  • the present invention provides a method of modulation of a defence response in a plant, the method including causing or allowing expression of nucleic acid according to the invention within cells of the plant.
  • it will be desirable to promote the defence response and this may be achieved by allowing Rarl gene function.
  • under-expression of endogenous Rarl gene may be achieved using anti-sense technology or "sense regulation".
  • anti-sense genes or partial gene sequences to down-regulate gene expression is now well-established.
  • Double-stranded DNA is placed under the control of a promoter in a "reverse orientation" such that transcription of the "anti-sense” strand of the DNA yields RNA which is complementary to normal mRNA transcribed from the "sense" strand of the target gene.
  • the complementary anti-sense RNA sequence is thought then to bind with mRNA to form a duplex, inhibiting translation of the endogenous mRNA from the target gene into protein. Whether or not this is the actual mode of action is still uncertain. However, it is established fact that the technique works.
  • Antisense technology is also reviewed in Bourque, 1995, and Flavell, 1994. Antisense constructs may involve 3'end or 5'end sequences of Rarl or homologues. In cases where several Rarl homologues exist in a plant species, the involvement of 5'- and 3 '-end untranslated sequences in the antisense constructs will enhance specificity of silencing.
  • Constructs may be expressed using the natural promoter, by a constitutively expressed promotor such as the CaMV 35S promotor, by a tissue-specific or cell-type specific promoter, or by a promoter that can be activated by an external signal or agent.
  • the CaMV 35S promoter but also the rice actinl and maize ubiquitin promoters have been shown to give high levels of reporter gene expression in rice (Fujimoto et al . , (1993) Bio/Technology 11, 1151-1155; Zhang, et al . , (1991) Plant Cell 3, 1155-1165; Cornejo et al . , (1993) Plant Molecular Biology 23, 567-581) .
  • the complete sequence corresponding to the coding sequence in reverse orientation need not be used.
  • fragments of sufficient length may be used. It is a routine matter for the person skilled in the art to screen fragments of various sizes and from various parts of the coding sequence to optimise the level of anti -sense inhibition. It may be advantageous to include the initiating methionine ATG codon, and perhaps one or more nucleotides upstream of the initiating codon.
  • a suitable fragment may have about 14-23 nucleotides, e.g. about 15, 16 or 17.
  • the present invention also provides a method of downwardly modulating Rarl expression in a plant, the method including causing or allowing anti-sense transcription from nucleic acid according to the invention within cells of the plant.
  • Rarl down-regulation may reduce a defence response. This may be appropriate in certain circumstances e.g. as an analytical or experimental approach.
  • nucleic acid including a nucleotide sequence complementary to a coding sequence of an Rarl gene (i.e. including homologues), or a fragment of a said coding sequence suitable for use in anti-sense regulation of expression, is provided.
  • This may be DNA and under control of an appropriate regulatory sequence for anti- sense transcription in cells of interest.
  • Suitable fragments may be about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650 or 700 nucleotides in length.
  • the present invention also provides a method of downwardly modulating Rarl function in a plant, the method including causing or allowing expression from nucleic acid according to the invention within cells of the plant to suppress endogenous Rarl expression.
  • Modified versions of Rarl may be used to down-regulate endogenous Rarl function. For example mutants, variants, derivatives etc., may be employed.
  • Reduction of Rarl wild type activity may be achieved by using ribozymes, such as replication ribozymes, e.g. of the hammerhead class (Haseloff and Gerlach, 1988, Nature 334 : 585-591; Feyter et al . Mol . , 1996, Gen . Genet . 250: 329- 338) .
  • ribozymes such as replication ribozymes, e.g. of the hammerhead class (Haseloff and Gerlach, 1988, Nature 334 : 585-591; Feyter et al . Mol . , 1996, Gen . Genet . 250: 329- 338) .
  • transposon mutagenesis Another way to reduce Rarl function in a plant employs transposon mutagenesis (reviewed by Osborne et al . , (1995) Current Opinion in Cell Biology 7, 406-413) .
  • Inactivation of genes has been demonstrated via a 'targeted tagging' approach using either endogenous mobile elements or heterologous cloned transposons which retain their mobility in alien genomes.
  • .Rarl alleles carrying any insertion of known sequence could be identified by using PCR primers with binding specificities both in the insertion sequence and the Rarl homologue. Two-element systems' could be used to stabilize the transposon within inactivated alleles.
  • a T-DNA is constructed bearing a non- autonomous transposon containing selectable or screenable marker gene inserted into an excision marker. Plants bearing these T-DNAs are crossed to plants bearing a second T-DNA expressing transposase function. Hybrids are double-selected for excision and for the marker within the transposon yielding F 2 plants with transposed elements.
  • Figure 1 shows the nucleotide and deduced amino acid sequences of the barley Rarl cDNA.
  • the nucleotide and the deduced amino acid sequence are based on the combined data of RT-PCR and RACE obtained from experiments using RNA of cultivar Ingrid Rarl .
  • the stop codon is marked by an asterisk and the detected termini of RACE products are indicated by arrows above the sequence.
  • Figure 2 illustrates the Rarl gene structure.
  • the structure of the barley Rarl gene is given schematically. Exons are highlighted by black boxes. Positions of introns and exons were identified by comparison of RT-PCR products with genomic sequences. Positions of mutational events are indicated for mutants rarl -1 and rarl -2.
  • Figure 3 shows an alignment of deduced peptide sequences in genes from various species indicating relatedness to barley Rarl. Regions of homology are highlighted in black (identity) , dark grey (highly conservative exchange) or light grey (conservative exchange) . Sequence data were analyzed with the Genetics Computer Group, Wisconsin Program, version 8 (GCG; Devereux, 1984) . Display of aligned deduced amino acid sequences were carried out by using the "prettybox" option in the extended GCG software. Numbers on the left indicate GenBank accession mumbers of each peptide sequence.
  • Figure 4 shows 10,000 nucleotides of the Rarl genomic gene sequence, including coding exons and introns .
  • the rarl -1 and rarl -2 mutations are marked.
  • Underlined sequences represent Rarl exon sequences and nucleotides in bold represent 5' and 3' consensus splice sequences.
  • Figure 5A shows the amino acid sequence of a N-terminal fragment of the Rarl polypeptide of Figure 1.
  • Figure 5B shows a nucleotide sequence encoding the Rarl polypeptide fragment of Figure 5A.
  • Figure 5C shows the amino acid sequence of a C-terminal fragment of the Rarl polypeptide of Figure 1.
  • Figure 5D shows a nucleotide sequence encoding the Rarl polypeptide fragment of Figure 5C.
  • Figure 6 shows the amino acid sequence of fragments and domains I, II and III of the Barley Rarl protein, representing particular aspects of the present invention.
  • Figure 7 shows nucleotide sequences encoding the fragments and domains I, II and III of Figure 6, polynucleotides with these sequences, and polynucleotides comprising these sequences, representing further aspects of the present invention.
  • Figure 8A shows AtRarl cDNA sequence, including coding sequence .
  • Figure 8B shows the AtRarl protein sequence (also shown in Figure 3 as ab010074) .
  • Figure 8C shows the encoding nucleotide sequence for an AtRarl protein N-terminal fragment.
  • Figure 8D shows the ArRarl protein N-terminal fragment encoded by the nucleotide sequence of Figure 8D.
  • Figure 8E shows the encoding nucleotide sequence for an AtRarl protein internal fragment .
  • Figure 8F shows the ArRarl protein internal fragment encoded by the nucleotide sequence of Figure 8E.
  • Figure 8G shows the encoding nucleotide sequence for an AtRarl protein C-terminal fragment.
  • Figure 8H shows the ArRarl protein C-terminal fragment encoded by the nucleotide sequence of Figure 8G.
  • Figure 9 shows alignment of various "CHORD” sequences ("Cysteine and Histidine Rich Domain”) and a consensus sequence .
  • a previous low resolution interval mapping procedure located Rarl on barley chromosome 2, flanked by RFLP loci CMWG694 and MWG503 within a 5 cM interval (Freialdenhoven et al . 1994) . Because 1 cM in barley corresponds to approximately 3 Mb we decided as a first step towards the isolation of Rarl to establish a local high resolution genetic map. We aimed at a resolution of approximately 0.01 cM which corresponds to an average physical distance of 30 kb.
  • the CAPS markers MWG876, MWG892 and MWG2123 were integrated into the genetic map.
  • a phenotypic screen was used to analyse 1040 plants for recombination events between Ant2 and Rarl .
  • the observed recombinants were used to reveal that MWG892 maps distal in relation to Rarl .
  • a subsequent CAPS-based recombinant screen of 1063 additional plants was performed in the marker interval MWG892 - cMWG694 . Analysis of the observed recombinants positioned MWG876 proximal in relation to Rarl .
  • RFLP markers MWG503 and CMWG694 which define an approximately 5 cM interval containing Rarl (Freialdenhoven et al . 1994) were sequenced, oligonucleotides for amplification of the corresponding loci were derived and polymorphisms between the susceptible (rarl-1 , rarl-2 ) and resistant parents (Mla-12 BC Ingrid, Mla-12 BC Pallas, Mla-12 BC Siri) were determined. This involved display on ethidium bromide stained 2.5% agarose gel of restriction enzyme digested amplification products using M82, MlOO, Mla -12 BC Ingrid, Mla-12 BC Pallas and Mla -12 BC Siri as template DNA. Amplification and t
  • CAPS markers corresponded to RFLP loci MWG87, MWG503, CMWG694, MWG876, and MWG2123.
  • PCR- primers for locus MWG892 enabled allele-specific discrimination of PCR products without subsequent restriction digestion. Minor bands were due to incomplete Bell digests of the PCR amplicons.
  • RFLP markers which map close to the above mentioned RFLP loci from a general RFLP map (MWG876, MWG892 and MWG2123 [Graner, 1991] .
  • MWG876, MWG892 and MWG2123 [Graner, 1991] .
  • Each of these RFLPs was converted to a cleavable amplifiable polymorphic sequence (CAPS) and was mapped relative to Rarl based on a population of 50 segregants .
  • CPS cleavable amplifiable polymorphic sequence
  • Cultivar Sultan-5 (Mla-12, Rarl) from which both Rarl mutants (rarl-1, rarl-2 ) are derived, contains an anthocyanin pigmentation deficiency (ant2) whereas the three resistant Mla-12 BC lines used for mapping (Mla-12 BC Ingrid, Mla-12 BC Pallas, Mla-12 BC Siri) carry the Ant2 wild type allele.
  • the Ant2 locus was previously shown to map at a distance of approximately 0.5 cM proximal to Rarl (Freialdenhoven et al . 1994) .
  • MWG87, MWG876 and Rarl may indicate small physical distances between these loci but could also be a result of a low recombination frequency in this genomic segment.
  • the susceptible pool (rarl-2 / rarl-2 ) contained three individuals with a recombination between cMWG694 and Rarl , four individuals with a recombination between MWG892 and Rarl and three susceptible individuals without a recombination event in the investigated marker interval.
  • the selection of recombinants for the resistant pool was based on DNA markers only. By using plants which show the allelic pattern of the resistant parent for CMWG694 and MWG892 we could ensure homozygosity in the corresponding genetic interval. Therefore linked AFLP markers are expected in trans and cis .
  • the genome-wide frequency of AFLP-polymorphisms between MlOO and Mla-12 BC Ingrid was found to be 7%.
  • Each AFLP primer combination displayed, on average, 100 DNA fragments.
  • the small amount of identified AFLP markers linked to Rarl is certainly influenced by the way we assembled the DNA pools. It may also indicate that the small genetic interval in which we searched for DNA markers is physically not excessively large. To obtain more precise estimates on the relationship of genetic and physical distances, we performed PFGE Southern analysis in combination with rare cutting restriction enzymes and RFLP probes linked to Rarl .
  • Fragment sizes after restriction with seven different rare cutting restriction endonucleases were determined using the cosegregating probe MWG87 and flanking probes MWG892 and MWG876. The analysis revealed a single co-migrating lul restriction fragments hybridising to MWG87 and MWG876 (Table 1) . This may indicate a maximal physical distance of 550 kb between MWG876 and MWG87. Fragments of common size were also detected using the probe/restriction enzyme combinations MWG876/NotI , Sail and Smal (90 kb) and MWG87/Sf " iI and Smal (100 kb) . These fragments of common size using one probe and different endonucleases are possibly caused by a clustering of restriction sites which has been reported before in vertebrates (Bickmore et al . 1992; Larsen et al. 1992) .
  • YAC end Y113L gave rise to amplification products in YAC Y73 and Y113.
  • the length of the PCR product in Y73 was different from the expected size which was detected in Y113. Therefore we concluded that the locus corresponding to Y113L was not present in YAC Y73.
  • All other YAC-end specific primers detected clear absence/presence polymorphisms on the different YAC clones and did not amplify fragments in Yl or Y2 , indicating their suitability for YAC contig analysis.
  • each end probe should detect the yeast clone it was derived from plus any YAC covering this area. For two YAC termini, marking the ends of the contig, only the yeast clone they were derived from should be detected. These end probes define now the termini of the YAC contig. Since we determined four YAC ends which are not amplified in any YAC, but the one they are derived from, at least two of the four YAC ends must be derived from chimeric YACs. However based on this information, it remained uncertain which two of the four YACs are chimeric. Genetic mapping of the YAC ends could resolve this lack of clarity but is only possible if the end probes are single or low copy markers.
  • oligonucleotides corresponding to YAC termini Y18L, Y18R, Y30L, Y30R, Y31R, Y73R and Y113R resulted in the amplification of uniformly sized fragments.
  • cloning and sequencing of the PCR products revealed that the oligonucleotides corresponding to Y30L and Y73L generated at least three different amplicons which show about 5% sequence divergence. This indicated that primers corresponding to Y30L and Y73L recognise multiple loci with high degree of sequence similarity.
  • sequence analysis was used for the selection of endonucleases recognising nucleotide stretches which are polymorphic between the three characterised subclones and the YAC-end derived sequence. Restriction digest of the PCR products with these diagnostic endonucleases resulted in a more complex banding pattern than predicted for an amplicon derived from a single locus of known sequence. Therefore endonuclease based analysis of the PCR products from genomic DNA confirmed heterogeneity of the amplification products corresponding to Y30L and Y73L and may be in general a useful tool to determine if a certain marker detects a single copy locus . Sequence analysis of the subclones corresponding to YAC termini Y18L, Y18R, Y30R, Y31R and Y113R indicated that these amplicons are homogenous.
  • the diteleosomic wheat/barley addition lines facilitate a rapid assignment of a given barley sequence to its corresponding chromosome arm if barley specific signals can be discriminated from wheat specific signals.
  • YAC end Y113R could not be mapped since the primers derived from cultivar Ingrid did not amplify a fragment from cultivar Betzes, the barley DNA donor for the addition lines. Similarly the YAC terminus Y30R could not be assigned because it generated fragments of identical size in wheat and barley.
  • the wheat/barley diteleosomic addition lines facilitate identification of chimeric YACs but high-resolution genetic mapping is necessary to define the position of the YAC ends in relation to the target locus.
  • a prerequisite for genetic mapping of the YAC termini is a sequence polymorphism between the parental genotypes of the mapping population.
  • Oligonucleotides corresponding to Y30R gave rise to amplification products of different size in the resistant (Rarl) and susceptible (rarl) parents whereas the primer pair corresponding to Y113R amplified DNA fragments only in each of the resistant parents.
  • PCR products derived from the resistant and susceptible parents for the marker loci Y18L and Y18R were analysed for DNA polymorphisms by direct sequencing. Comparative sequence analysis revealed a polymorphic Hinfl site in the case of Y18R whereas in Y18L, no DNA polymorphism was detected over about 2.7 kb. A copy of a BARE-1 retrotransposon within the Y18L sequence made it impossible to further extend this sequence by IPCR to search for polymorphisms. Genetic mapping of the polymorphic YAC ends positioned Y30R and Y113R proximal to Rarl , separated by eleven and three recombinants respectively from the target locus. Marker Y18R was found to cosegregate with Rarl .
  • YAC Y18 is likely to be the only YAC containing a non-chimeric insert which is colinear to the source DNA, since both termini map to chromosome 2HL.
  • YAC Y30 has probably undergone a rearrangement leading to an internal deletion including the marker Y113R. This conclusion is based on (i) the genetic mapping of Y113R between MWG87 and Y30R and (ii) absence of marker Y113R in YAC Y30 (Table 2) .
  • the genomic area containing Rarl is genetically delimited by Y113R (proximal) and MWG876 (distal) . Since the presented YAC contig covers this interval physically by YAC clones Y18 and Y113 in proximal (two fold redundancy) , and YAC clones Y30 and Y31 in distal orientation (two fold redundancy), we have physically delimited the Rarl locus.
  • BACs Five BACs, derived from YACs Y30 and Y18, were initially isolated with CAPS MWG87, cosegregating with Rarl (BAC 12, BAC 1J6, BAC 4C20, BAC 1G12 , and BAC 3H6) .
  • PCR primers for marker Y113R were used to isolate BAC 1H1. Insert sizes of the identified BACs were determined by PFGE. End fragments of each BAC insert were isolated by inverse PCR and subsequently sequenced. Based on terminal sequences of
  • BAC 4C20 we derived a new co-dominant DNA marker, designated EDDA (Table 4) , detecting a sequence polymorphism between the parental genotypes of the mapping population. Analysis of the four recombinants within interval MWG876 - Y113R, positioned EDDA proximal to Rarl . Since BAC 4C20 contains each of the three loci MWG87, Y18R, and EDDA, we have physically delimited Rarl in direction to the centromere on a single BAC clone.
  • BAC 12, BAC 3H6, and BAC 1B2 were employed to derive markers OKI114, OK3236, and OK5558, respectively (Table 3) .
  • the co-dominant marker OK1114 was found to cosegregate with Rarl by inspection of genomic DNA derived from the four recombinants within target interval MWG876 - Y113R.
  • Markers OK3236 and OK5558 detected polymorphisms between the parental genotypes Sultan5/Mla- 12 BC Pallas and Sultan5/ la-12 BC Ingrid but we failed to detect a polymorphism for this locus between Sultan5/Mla- 12 BC Siri.
  • Rarl was physically delimited on the BAC level in centromeric orientation and the identification of a minimal cosegregating interval bordered by markers Y18R and OK5558.
  • Intervals II and 12 are sequence related to each other (59% nucleotide identity) and were identified by the same class of ESTs in the databases (Table 5) , each showing similarity to aquaporin genes [Maurel , 1997] .
  • Interval 13 shows high sequence similarity ro rice EST C28356 and may represent another coding region in the 66 kb strech.
  • RT-PCR reverse transcriptase-polymerse chain reactions
  • the G->A DNA substitution identified in genotype rarl - 1 Sultan5 results in a Cys 24 ->Tyr substitution in the putative 25.5 kDa protein (Cys 24 represents one of the few invariant amino acids in Rarl homologous proteins; see below) .
  • the G->A DNA substitution identified in genotype rarl -2 Sultan5 disrupts the 3' splice site consensus sequence of intron 2.
  • the G nucleotide of the splice site consensus is known to be essential for effective splicing of primary mRNA transcripts in both plant and mammalian species [Goodall, 1991] .
  • RT-PCR analysis of the rarl -2 genotype revealed that the mutation leads to utilisation of a cryptic splice site in exon 3, a phenomenon documented in numerous human herditary diseases caused by point mutations [Krawczak, 1992] [Brown, 1996] .
  • Use of this cryptic splice site leads to a shift of the reading frame, creating a new stop codon, and consequently a truncation of the deduced 25.5 kDa protein.
  • domain I, II, and III - Figure 6 A close inspection of the Rarl protein sequence and a comparison to the rice and Arabidopsis homologues reveals a striking tripartite structure (designated domain I, II, and III - Figure 6) .
  • domain I and III each approximately 60 aa long and located close to the amino- and carboxy-terminal ends of Rarl respectively, are structurally related to each other. Remarkable is a strictly conserved pattern of cysteine and histidine residues in domains I and III.
  • the domain signature is not only conserved among plant Rarl homologues but it is also found in each of the other related peptide stretches identified in proteins from Aspergillus, Drosophila, Caenorhabdi tis , mouse, and man.
  • CHORD novel protein domain
  • the example in domain I of Rarl being termed “CHORDl”
  • domain III being "CHORD2”
  • CHORD contains few other invariant amino acids, Gly 23 , Phe 47 , and Trp 54 as well as a negatively charged residue in position 49 and a positively charged residue at position 52 (numbering refers to the amino terminal CHORD domain in barley Rarl) .
  • CHORD conserved strings of Cysteine and Histidine residues in intracellular protein domains have been frequently shown to be involved in binding zinc ions.
  • the pattern of these residues in CHORD is distinct from any previously described zinc-binding domain in which zinc ions have a structural role to stabilize small, autonomously folding and functional protein domains (e.g. the TFIIIa zinc finger, the GAL4 zinc finger, the zinc binding domain in the oestrogen receptor, the LIM domain, the RING finger domain, and the GATA-1 finger domain) .
  • the CHORD domain (e.g. CHORDl and CHORD2) can be signified as
  • CHORD domain may conform to the formula: 11
  • C, G, F and W are the single letter code for Cys, Gly, Phe and Trp, respectively,
  • a 1 is an aromatic amino acid, and may be selected from Phe,
  • a 2 is a negatively charged residue, and may be selected from
  • a 3 is a positively charged residue, and may be selected from Arg, His and Lys, y x is H or any amino acid, and is preferably His or Arg, and X may be any amino acid (with the numbers indicating the number of amino acids) , subject to the structural constraints on the spatial relationship of the cysteines and histidines required for zinc binding.
  • Domain III in plant Rarl-like proteins appears to contain another set of cysteine and histidine residues providing a domain according to the present invention: C-x 2 -C-x 5 -C-x 2 -H.
  • Arabidopsis thaliana NPR1 gene a key regulator in systemic aquired resistance, resulted in complete resistance to the pathogens Peronospora parasi tica and Pseudomonas syringae [Cao, 1998] , providing indication that modulating steady state levels of Rarl mRNA or protein may be used to alter speed and pathogen spectrum of the resistance response.
  • Redirecting Rarl expression by fusing the gene to promoters from pathogenesis-related genes may also be used to broaden the spectrum of Rarl mediated pathogen resistance. This approach may be particularly attractive in combination with the expression of derivatives of the Rarl protein.
  • modified versions of the Rarl protein may be identified which decouple its activation from R genes and retain their activation of downstream responses (PR gene activation, HR) .
  • the identified tripartite domain structure of the plant Rarl proteins may serve in guiding these experiments .
  • the Mla -12 BC line in cultivar Ingrid was generated through seven backcrosses with H. vulgare cv Ingrid followed by at least seven selfings. Each of the mutants M82 and MlOO were pollinated with pollen derived from the Mla -12 BC line cultivars, F 1 plants from each cross were grown to maturity providing the various segregating F 2 populations.
  • Tests for Resistance Tests for resistance were carried out as described in Freialdenhoven et al . 1994.
  • the phenotype of the recombinants was determined after selfing and subsequent inoculation experiments in F 3 and F 4 families comprising at least 25 individuals.
  • F 3 individuals were tested by cleavable amplifiable polymorphic sequence analysis (CAPS) to identify homozygous recombinants. These plants were again selfed and subjected to resistance tests in F 4 families. Plants were scored for resistance/susceptibility seven days after inoculation.
  • Genomic DNA for CAPS and AFLP analysis was isolated according to Stewart and Via (1993) .
  • Primer PCR conditions and the respective restriction enzymes used for CAPS marker display are shown in Table 1.
  • CAPS analysis was performed in a volume of 20 ⁇ l (100 pmole of each primer, 200 ⁇ M dNTPs, 10 mM Tris-HCl pH 8.3 , 2 mM MgCl 2 , 50 mM KC1 2 , 0.5 U Taq Polymerase (Boehringer) using 50 ng of barley genomic template DNA.
  • the digested PCR products were subsequently size-fractionated on 2% agarose gels.
  • AFLP analysis (Vos et al .
  • Plant DNA for PCR-based analysis was extracted according to (Stewart and Via 1993) .
  • Primer and PCR conditions for YAC end specific markers are listed in Table 4.
  • PCR was performed in a volume of 20 ⁇ l (100 pmole of each primer, 200 ⁇ M dNTPs, 10 mM Tris-HCl pH 8.3, 2 mM MgCl 2 , 50 mM KC1 2 , 0.5 U Taq Polymerase (Boehringer Mannheim, Mannheim, Germany) using 200/50 ng of wheat/barley genomic template DNA.
  • Amplicons corresponding to the different YAC ends were cloned into pGEM-T vector (Promega, Southampton, United Kingdom) and three independent clones of each PCR product were subjected to dye terminator cycle sequencing (Perkin Elmer) .
  • yeast chromosomes Separation of yeast chromosomes was performed by a 1.2% agarose gel (SeakemTM LE; FMC BioProducts, Rockland, ME, USA) in an LKB PulsaphorTM apparatus (Pharmacia Biotech, Uppsala, Sweden) at 180 V with pulse times from 10- 80 s (linear interpolation) for 30 h in 0.5x TBE (50 mM Tris- HCI, 50 mM boric acid, 1 mM EDTA, pH 8.3) at 12 °C. MWG87 was used subsequently as a probe for Southern hybridisation as described in Lahaye et al . (1996) to determine the size of the YAC inserts .
  • Seeds of Hordeum vulgare cv. Golden Promise are sterilized by incubation in 70% ethanol for one min, washing three times in Milli-Q water followed by incubation in 1.5% sodium hypochlorite for 10 min and 5 times washing in sterile Milli- Q water. Seeds are sown in magentas (15 seeds/sample) containing 2 cm vermiculite supplied with 30 ml 1/2 -strength MS basal medium (Sigma) supplemented with 2% sucrose, and cultured at 22 C (16 h light/8h darkness) .
  • the transformation process uses a particle inflow gun (PIG) (Vain et al . 1992).
  • PIG particle inflow gun
  • the constructs are precipitated onto gold particles (l ⁇ m, Bio Rad) according to the method of Klein et al . 1988 introducing l ⁇ g of Quiagen-purified plasmid per bombardment.
  • the plant material is placed 9cm below the particle outlet and covered with a steel grid with 0.4 mm pore size. Bombardment conditions are: acceleration of the particle with 2735 mbar Helium gas at an air pressure of 100 mbar.
  • approx. 4ml of sterile 1/2 strength MS basal medium supplied with 3% sucrose and ImM benzimidazol, is added to the sample which is then incubated at 24°C in the dark for 24h.
  • PR genes are known to be activated at high levels surrounding the sites of attempted pathogen attack.
  • Cell death activating Rar-1 derivatives are fused to 2 kb promoter sequences of barley genes HvPRl-a and HvPRl-b (Bryngelsson et al . 1994) . These genes are known to be activated in leaf tissue in response to attack from different pathogens including powdery mildew, Drechslera teres, and Puccinia hordei (Reiss and Bryngelsson 1996) .
  • HvPRl-a and HvPRl-b promoters to cell death activating Rarl derivatives as provided herein are cloned into vector pAHC25 (Wan and Lemaux 1994) by replacing both the uidA reporter gene and the maize ubiquitin promoter of pAHC25, following standard cloning procedures (Sambrook et al . 1989).
  • Transgenic lines are tested for broad spectrum resistance following inoculations with different isolates of powdery mildew, Puccinia hordei , and Drechslera teres spores. Plants displaying resistance to the described isolates are observed.
  • Rarl constructs activating host cell death in the transient assay are selected for further modification in transgenic plants.
  • Preparation of plant material is carried out as described above for barley using surface-sterilized Arabidopsis (ecotype Columbia) and tomato seeds (cultivar Moneymaker) .
  • Leaves of 4 week old plants are infiltrated with Agrobacterium strain C58 containing p35S-Rarl constructs in which Rarl derivatives are driven by the 35S CaMV promoter in a T-DNA vector, pBIN19 (Bevan, 1984) .
  • Rarl constructs activating host cell death in the transient assay are selected for further modification in transgenic plants .
  • glucocorticoid-mediated transcriptional induction system (Aoyama and Chua, 1997, Plant Journal 11 , 605-612.) is employed in inducible over-expression of full length and truncated AtRarl gene derivatives in Arabidopsis .
  • the vector pTA231 (The Rockefeller University, New York, USA) is modified by inserting the AtRarl sequence of interest selected from those shown in Figures 8A to 8H, i.e. the whole AtRarl gene, a sequence encoding an N-terminal portion, an internal portion, or a C-terminal portion thereof, using the Xhol and Spel cloning sites.
  • Primers used to amplify AtRarl gene fragments are:
  • Arabidopsis plants are transformed with Agrobacterium strain C58 containing above constructs by a standard Arabidopsis transformation protocol (Clough and Bent, 1998, Plant Journal 16, 735-743) .
  • a plus sign (+) indicates that a YAC was positive by PCR analysis for the respective marker listed above. Absence of a marker is indicated by a minus sign (-).
  • CMWG694 5'-AGTATCAGATGCTACCATGCCTGG 94°C , 10 s Haelll 5 '-CTCTGGAGGAGCCGAGTGTC AGC 60°C , 20 s
  • MWG892 5'-GGAATCTTCCAGTGGGCTGGATGAG 94°C 10 s 5'-CAACCGGCCACTAGGCGTAAAGG 60°C, 20 s

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Abstract

Gène de signalisation de la résistance aux maladies des plantes, Rar1, provenant de l'orge, du riz, d'Arabidopsis, et homologues provenant d'autres espèces. L'acide nucléique et les polypeptides codés sont utiles pour moduler chez les végétaux la voie de signalisation qui aboutit soit à une réponse de défense aux agents pathogènes des plantes et/ou à la mort cellulaire, soit à la résistance aux agents pathogènes due à l'interaction des produits du gène R avec des protéines pathogènes Avr.
PCT/GB1999/002590 1998-08-06 1999-08-06 Gene de signalisation de la resistance aux maladies des plantes: materiaux et methodes Ceased WO2000008160A2 (fr)

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AU54288/99A AU760571B2 (en) 1998-08-06 1999-08-06 A plant disease resistance signalling gene: materials and methods relating thereto
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JP2000563785A JP2002524044A (ja) 1998-08-06 1999-08-06 植物病耐性シグナル伝達遺伝子：それに関する材料及び方法
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6627796B2 (en) 2000-02-09 2003-09-30 Plant Bioscience Limited Maize Rar1 polynucleotides and methods of use
WO2003048339A3 (fr) * 2001-11-30 2005-04-21 Syngenta Participations Ag Molecules d'acides nucleiques du riz codant pour des proteines rar1 de resistance aux maladies et leurs utilisations
US7977087B2 (en) 2004-03-31 2011-07-12 Japan Science And Technology Agency Detection instrument with the use of polynucleotides mapped on barley chromosome
EP3344776A4 (fr) * 2015-09-04 2019-09-04 Synthetic Genomics, Inc. Microorganismes modifiés pour améliorer la productivité

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Publication number Priority date Publication date Assignee Title
CN100549028C (zh) * 2006-01-19 2009-10-14 中国农业科学院作物科学研究所 植物抗病相关蛋白rar1及其编码基因与应用
CN102257144B (zh) * 2008-11-10 2015-04-01 双刃基金会 病原体可诱导的启动子及其在增强植物的疾病抗性中的用途

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EP0917536B8 (fr) * 1996-07-29 2009-01-07 Keygene N.V. Polynucleotide et son utilisation pour moduler une reponse de defense dans des plantes
CZ39799A3 (cs) * 1996-08-09 1999-07-14 The General Hospital Corporation NPR geny získané resistence a jejich použití
US6166295A (en) * 1996-11-22 2000-12-26 The Regents Of The University Of California Composition and method for plant pathogen resistance

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6627796B2 (en) 2000-02-09 2003-09-30 Plant Bioscience Limited Maize Rar1 polynucleotides and methods of use
WO2003048339A3 (fr) * 2001-11-30 2005-04-21 Syngenta Participations Ag Molecules d'acides nucleiques du riz codant pour des proteines rar1 de resistance aux maladies et leurs utilisations
US6956115B2 (en) 2001-11-30 2005-10-18 Syngenta Participations Ag Nucleic acid molecules from rice encoding RAR1 disease resistance proteins and uses thereof
US7098378B2 (en) 2001-11-30 2006-08-29 Syngenta Participations Ag Transgenic plants compromising nucleic acid molecules encoding RAR1 disease resistance proteins and uses thereof
US7977087B2 (en) 2004-03-31 2011-07-12 Japan Science And Technology Agency Detection instrument with the use of polynucleotides mapped on barley chromosome
EP3344776A4 (fr) * 2015-09-04 2019-09-04 Synthetic Genomics, Inc. Microorganismes modifiés pour améliorer la productivité
US10683514B2 (en) 2015-09-04 2020-06-16 Synthetic Genomics, Inc. Microorganisms engineered for increased productivity

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