JP2002524044A - Plant disease resistance signaling gene: Materials and methods related thereto - Google Patents

Abstract

  (57) [Summary] Plant disease resistance signaling gene Rar1 from barley, rice, Arabidopsis, and homologues from many species. Nucleic acids and encoded polypeptides are useful for modulating plant signaling pathways, and the interaction of the R gene product with the pathogen Avr protein results in a plant pathogen defense response and / or killing, or pathogen resistance .

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a plant disease resistance signaling gene, a substance and a method related thereto. In particular, the invention relates to the Rar1 gene of barley and its homologs from other species.

[0002] Plant resistance to pathogens is responsible for a series of defense-related responses, such as the production of antimicrobial compounds, activation of pathogenesis-related (PR) genes, cross-linking of plant cell walls, and production of reactive oxygen species. It is known to be associated with induction (Dixon and Lamb 1990; H
ammond-Kosack and Jones 1996). In most cases, resistance is related to the response of host cell death at the site of the attempted infection, called the hypersensitivity response (HR). However, in using biochemical and physiological techniques to block the growth of invasive factors, it has proven difficult to imply a particular response.

[0003] In contrast, genetic studies have defined a locus, the R gene, which plays a major role in responding to microbial pathogens carrying the corresponding non-pathogenic determinant (Flor, 1971). Thus, knowledge of these resistance gene products and their significant pathways facilitates deeper insights into plant defense mechanisms. In recent years, a number of R genes have been isolated (Martin et al., 1993; Bent et al., 1994; Jones).
Et al., 1994; Mindrinos et al., 1994; Grant et al., 1995; Lawrence et al., 1995; Song et al., 19
95; Anderson et al., 1997; Cai et al., 1997; Ori et al., 1997; Yoshimura et al., 1998).

[0004] Most R genes isolated from plants encode variable stretches of leucine-rich repeats and putative nucleotide binding sites, or encode leucine-rich repeat domains, but do not bind to nucleotide binding. Do not code part, 2
Enter one class. Each of the third and fourth classes has so far only one representative and encodes a protein with a leucine-rich repeat and a serine-threonine protein kinase domain, or only a protein. However, little is known about the exact actions of the R genes and the downstream pathways that they affect.

[0005] The complexity of plant defense responses suggests that other components, excluding the R gene, are involved in the resistance response. The first genetic evidence for additional components in breed-specific resistance is that the identification of Rar1 and Rar2 (these two components are required for activation of the R gene M1a-12) will result in barley-powdery powdery mildew (powdery ) (Previously designated Nar-1 and Nar-2; Torp and Jorgensen)
Jorgensen, 1988; Freialdenhoven et al., 1994). Subsequent studies of other plant pathogen interactions have revealed similar observations (Hammond-Kosack et al., 199).
4; Salmeron et al., 1994; Century et al., 1995; Parker et al., 1996), and two of these components have recently been described as a simple dicot model species Arabidopsis thaliana (A
rabidopsis thaliana) (unpublished PCT / GB98 / 01406; Century)
Et al., 1997).

[0006] Both of these Arabidopsis genes, EDS1 and EDS2, are required for multigene factors for different pathogens, ie bacterial and fungal pathogens. NDR1 encodes a putative integral membrane protein with unknown biochemical function, whereas EDS1 is predicted to encode a putative novel plant lipase.

To date, the Mla loci on chromosome 1H (Mla-6, Mla-9, Mla-12, Mla-13, Mla
breed-specific resistance specificity of most of the powdery mildews tested, encoded by la-14, Mla-22, and Mla-23), and other loci (Mlat, Mlh, Mlk
Evidence shows that mutations in barley Rar1 suppress the resistance specificity against powdery mildew in Mlra, and Mlg) (Jorgensen, 1996; Peterhans
el et al., 1997). However, in some cases, no suppression of resistance gene function was observed in Mla-1, Mla-7 and mlo (Jorgensen, 1996; Peterhansel).
Et al., 1997).

[0008] Two chemically induced allelic mutations in Rar1 (designated rar1-1)
(Mutation M82) and rar1-2 (mutation M100) were characterized (Freialdenh
oven et al., 1994). Each of these two recessive alleles allows the powdery mildew pathogen to complete its lifespan in the presence of the aforementioned powdery mildew R gene. The epidermal single cell HR, a characteristic early event of the Mla-12 specific resistance response, is destroyed in the presence of rar1-1 or rar1-2. However, at a later stage, the susceptible infectious phenotype in the presence of rar1-1 or rar1-2 is easily identifiable. Low aerial mycelium and low sporulation are observed in the presence of the former defective allele, and the site of infection is often surrounded by necrotic host tissue.

[0009] In contrast, the infectious phenotype in the presence of rar1-2 is rarely distinguishable from the fully diseased barley genotype. We have interpreted these findings as the rar1-1 allele retains residual gene activity and recently demonstrated that rar1-2 also retains it. The aforementioned genetic data provide strong evidence that Rar1 represents a convergent point in the signaling of powdery mildew resistance induced by multiple powdery mildew R genes.

[0010] A reliable biochemical test procedure that allows for the usual purification of the Rar1 protein has not been described in the literature. Primarily due to an unfavorable ratio of genetic and physical distance, and repetitive non-coding
Due to the high percentage of DNA sequences, map-based gene isolation from highly complex barley genomes (5.3 × 10 ⁹ bp / haploid genome; (Bennett and Smith 1991)) is a major experimental challenge Despite the fact that it conferred the invention, the present invention succeeded in cloning the Rar1 gene from barley using positional cloning. To date, only one successful case of map-based isolation of the barley gene has been reported (Beschges et al., 1997).

The present invention relates to the cloning of the Rar1 gene, its homologs and mutant alleles. In various aspects, the invention relates to nucleic acids that encode polypeptides having Rar1 function. "Rar1 function" is a signaling pathway that leads to cell death, preferably pathogen resistance, which is achieved by a plant pathogen defense response and / or direct or indirect interaction of the R gene product with the pathogen Avr protein. Gene and its polypeptide expression products are meant.

The term “Rar1 function” is used to mean a sequence that directs the Rar1 phenotype in a plant, and the term “Rar1 mutation function” or “rar1 function” suppresses or abolishes the Rar1 phenotype in a plant. It can be used to refer to the form of the Rar1 sequence. The rar1 mutant phenotype is characterized by reducing or abolishing pathogen resistance and / or plant pathogen defense responses.

[0013] Rar1 function and rar1 function can be determined by assessing the level of plant defense response and / or susceptibility to pathogens as described above;
Alternatively, it can be determined by other suitable alternatives known in the art and available to those skilled in the art. The test plant can be a monocot or dicot. Suitable monocotyledonous plants include barley, rice, wheat, corn or oats, especially barley. Suitable dicotyledonous plants include Arabidopsis, tobacco, tomatoes, Brassicas, potatoes and grapes.

A polynucleotide according to the invention can encode a polypeptide comprising the amino acid sequence shown in FIG. The coding sequence is a sequence shown to be included in FIG. 1 or a mutation, variant,
It can be a derivative or an allele. The sequence can differ from the shown sequence by changes that are one or more additions, insertions, deletions and substitutions of one or more nucleotides of the shown sequence.

Changes to the nucleotide sequence may or may not result in amino acid changes at the protein level, as determined by the genetic code. Thus, a nucleic acid according to the present invention can include a sequence that differs from the sequence shown in Figure 1 and still encode a polypeptide having the same amino acid sequence (ie, the coding sequence is "degenerately equivalent"). There can be).

The polynucleotide according to the invention is identified as an exon in FIG.
Alternatively, it can include more than one sequence. It is also clearly related to functional Rar1 polypeptides (eg, exhibiting Rar1 function).
A nucleotide encoding a polypeptide comprising an amino acid sequence that is immunologically cross-reactive with the Rar1 polypeptide or has a characteristic sequence motif common to the Rar1 polypeptide) but no longer has Rar1 function. Nucleic acid molecules containing sequences are included within the scope of the present invention. Thus, the present invention provides Rar1 mutations that do not promote plant pathogen defense responses or cell death, and / or pathogen resistance. Plants and plant cells carrying these mutations are susceptible to pathogen infection.

Thus, Rar1 mutations, variants, fragments, derivatives, alleles and homologs of both the resistance-generating and resistance-reducing types may have practical values depending on the situation . In the agronomic context, a major interest is the development of plant resistance to pathogens.

In particular, homologs of certain Rar1 sequences described herein (see, eg, FIG. 3),
And such homologous mutations, variants, fragments and derivatives are provided by the present invention (and the foregoing remarks regarding such mutations and others also apply to homologous mutations and others) ). Such homologs are readily obtained by the disclosure provided herein. Thus, the present invention also provides
Derived from FIG. 1 or FIG. 4, or can be obtained using the nucleotide sequence shown in FIG. 1 or FIG. 4, or using the amino acid sequence shown in FIG. 1 or FIG. The present invention extends to nucleic acid molecules that include a nucleic acid sequence encoding a Rar1 homolog, which can be obtained as follows.

The Rar1 homologue has homology at the nucleotide level to the nucleotide sequence of FIG. 1, or has a homology to the polypeptide of the amino acid sequence shown in FIG. 1, preferably at least about 50%, Or encodes a polypeptide that is 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%, or at least about 90% homologous be able to. More preferably, at least about 95%
Homology. (The determination of homologues at the amino acid level is further described below).

In certain embodiments, an allele, variant, derivative, mutant derivative, mutation or homolog of a polypeptide of a particular sequence has a slight overall homology to the particular amino acid sequence of FIG. Indicating about 20%, or about 25%, about 30%,
Or about 35%, about 40%, or about 45% homology. However, in functionally significant domains or regions, the amino acid homology can be very high. Putative functionally significant domains or regions can be identified using bioinformatics processes, including comparison of homologous sequences.

[0021] Functionally significant domains or regions of different polypeptides can be combined for expression from the encoding nucleic acid as a fusion protein. For example, certain advantageous or desirable properties of different homologs can be combined in a hybrid protein, and the resulting expression product can have Rar1 or Rar1 function and include fragments of various parent proteins. Fragments according to other aspects of the invention are shown in FIGS. 5A and 5C, and the coding nucleotide sequences are shown in FIGS. 5B and 5D, respectively.

Other domains and fragments of the invention are shown in FIG. 6 and the coding nucleotide sequence is shown in FIG. These domains and fragments can be used in various aspects and embodiments of the invention disclosed herein. Each nucleotide sequence in FIG. 7 represents another aspect of the invention, and polynucleotides comprising the sequences as shown can be used in various aspects and embodiments disclosed herein.

Using Rar1-derived oligonucleotide primers (as intrinsic or degenerate sequences), monocotyledonous and dicotyledonous plants, such as barley, wheat, corn, oats, rice, tomato, melon, cucurbitaceae (cucurbitaceae) Rar1 homologs can be isolated from a number of different plants, including Brassicaceae, capsicum, lettuce, grape, and ornamental plants.

Once the corresponding gene has been cloned, it can be expressed as an antisense construct to assess its importance in resistance to agronomically important diseases, such as the following: Puccin
ia hordei) (leaf rust disease), Linchosporium secaris (Rhynchosproium se)
calis) (pyretic rot), Pyrenophera teres (net blotch), Heterodera avene (Heterodera avenae) (barley cereal cyst nematode), Drechslera teres, powdery mildew And yellow dwarf virus (eg, barley yellow dwarf virus).

Obtaining homologs is described below, but the nucleotide sequence information provided herein, or a portion thereof, can be used in a database search to find homologous sequences, and the expression product thereof is identified as Rar1 (or rar1) It should be pointed out briefly that functionality can be tested. They have the ability to complement the Rar1 (or rar1) phenotype in plants or can confer such a phenotype when expressed in plants. Thus, Rar1 cDNA or a portion thereof can be used as a bait in an interaction trap assay, for example, a yeast two-hybrid system, to isolate other previously unknown disease resistance signaling components. These represent other goals for manipulating pathways towards improved disease resistance.

By sequencing homologs, studying their expression patterns and examining the effects of altering their expression, genes can be obtained that perform functions similar to Rar1. Of course, mutations, variants, and alleles of these sequences fall within the scope of the invention in the same terms described above for the barley Rar1 gene. However, homologous sequences pre-existing on the database, e.g., the identified sequences included in FIG. 3, can be excluded from one or more aspects or embodiments of the invention, while one or more other It should be noted that

The homology between the homologs disclosed herein can be determined using, for example, oligonucleotides (eg, degenerate pools) or PCR primers designed on the basis of sequence conservation or further homologs. It can be used in the identification of Primers useful in the context of the present invention are "AtRar1 5 '" and "AtRar1 3'
And their sequences are described below.

According to another aspect, the invention provides a method for identifying or cloning a Rar1 homolog, for example, from a species other than barley, wherein the method comprises the steps shown in FIG. 1 or FIG. The nucleotide sequence derived from the sequence is used. For example, such a method provides a preparation of a plant cell nucleic acid and comprises a nucleotide sequence substantially as set forth herein or substantially complementary to the nucleotide sequence set forth herein, preferably a coding sequence ( For example, a nucleic acid molecule having a nucleotide sequence from within the sequence encoding the amino acid sequence shown in FIG. 1) is provided, and the nucleic acid molecule is hybridized to the gene or homolog in the preparation under the conditions described above. Contacting a nucleic acid in a preparation with the nucleic acid molecule and, if present, identifying the gene or homologue by its hybridization to the nucleic acid molecule can be included.

The target or candidate nucleic acid can be, for example, genomic DNA obtainable from an organism, monocot or dicot that is known or suspected of containing such nucleic acid. , CDNA or RNA (or a mixture of any of these, preferably as a library). Prior to the PCR to be performed, create a cDNA library using, for example, RT-PCR, or use phenol emulsion reassociation technology (Clarke et al. (1992) NAR 20, 1289-1292) for the genomic library. This can reduce the complexity of the nucleic acid library. Promising hybridizations can be identified and target / candidate nucleic acids can be isolated for further study and / or use.

[0030] Hybridization of a nucleic acid molecule to a Rar1 gene or homolog can be measured or identified indirectly, for example, using a nucleic acid amplification reaction, particularly the polymerase chain reaction (PCR). Preferably, two nucleic acid molecules having sequences exhibiting the characteristics of Rar1 are used because PCR requires the use of two primers to specifically amplify the target nucleic acid. However, when using RACE, only one such primer is required.

Alternatively, hybridization can be measured by probing with nucleic acids and identifying positive hybridization (under known techniques) under appropriately stringent conditions (amplification techniques, if necessary, For example, PC
R in combination). For probing, preferred conditions are those that are sufficiently stringent for the presence of a simple pattern, with small numbers of hybridizations identified as positive, which can be further studied. It is well known in the art to gradually increase the stringency of hybridization until only a few positive clones remain.

The binding of a probe to a target nucleic acid (eg, DNA) can be measured by various techniques known in the art. For example, the probe can be labeled with radiation, fluorescence or an enzyme. Other methods that do not label the probe include testing for restriction fragment length polymorphisms, amplification using PCR, RNase cleavage, and probing allele-specific oligonucleotides.

In probing, standard Southern blotting techniques can be used. For example, DNA can be extracted from cells and digested with different restriction enzymes. The restriction fragments are then separated by electrophoresis on an agarose gel, denatured and transferred to nitrocellulose filters. Label the labeled probe on the filter
The A fragment can be hybridized and binding determined. DNA for probing can be prepared from DNA preparations from cells by techniques such as reverse transcriptase-PCR.

Preliminary experiments can be performed by hybridizing various probes under low stringency conditions to Southern blots of restriction enzyme digested DNA. For probing, preferred conditions are those that are sufficiently stringent for the presence of a simple pattern, with small numbers of hybridizations identified as positive, which can be further studied. It is well known in the art to gradually increase the stringency of hybridization until only a few positive clones remain.

Although a large number of hybridizing fragments are obtained, suitable conditions will be achieved when background hybridization is low. Under these conditions, a nucleic acid library representative of the expressed sequence, for example,
You can search cDNA libraries. One skilled in the art can often use appropriate conditions for the desired stringency for selective hybridization, taking into account such factors as oligonucleotide length and base composition, temperature and other factors.

For example, at a temperature of about 37 ° C. or higher, a formamide concentration of less than about 50%,
Initially, the screening was performed under conditions comprising a medium to low salt (e.g., standard saline-citrate buffer ("SSC") = 0.15 M sodium chloride; 0.15 M sodium citrate; pH 7). Can be implemented.

Alternatively, a temperature of about 50 ° C. or higher and a high salt (eg, “SSPE” =
0.180 mM sodium chloride; 9 mM disodium hydrogen phosphate; 9 mM sodium dihydrogen phosphate; 1 mM sodium EDTA; pH 7.4). Preferably the screening is about
It is performed at 37 ° C., a formamide concentration of about 20%, and a salt concentration of about 5 × SSC, or at a temperature of about 50 ° C. and a salt concentration of about 2 × SSPE. Under these conditions, sequences with a substantial degree of homology (similarity, identity) with the probe sequence can be identified without the need for complete homology for the identification of stable hybrids.

Suitable conditions are, for example, 42% for the detection of sequences having about 80-90% identity.
Includes overnight hybridization in 0.25 M Na ₂ HPO ₄ , pH 7.2, 6.5% SDS, 10% dextran sulfate at 0 ° C., and final wash in 0.1 × SSC, 0.1% SDS at 55 ° C. . For the detection of sequences with greater than about 90% identity, suitable conditions are overnight at 65 ° C. in 0.25 M Na ₂ HPO ₄ , pH 7.2, 6.5% SDS, 10% dextran sulfate. Hybridization and 0.1 × SSC at 60 ° C., 0.
Includes final wash in 1% SDS.

In general, hybridization can be performed according to the method of Sambrook et al. (Below) using a hybridization solution comprising the following components: 5 × SSC (where “SSC” = 0.15M) Sodium chloride; 0.15 M sodium citrate; pH 7), 5 × Denhardt's reagent, 0.5-1.0% SDS, 100 μg / ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide . Hybridization is performed at 37-42 ° C for at least 6 hours. After hybridization, filters are washed as follows: (1) 5 minutes in 2 × SSC and 1% SDS at room temperature; (2) 2 × SSC and 0.1% S at room temperature
15 minutes in DS; (3) 30 minutes to 1 hour in 1 × SSC and 1% SDS at 37 ° C .; (4) 4
The solution is changed every 30 minutes for 2 hours in 1 × SSC and 1% SDS at 2-65 ° C.

A common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of specified sequence homology is as follows (Sanger et al., (1989)): Tm = 81.5 ° C + 16.6 Log [Na +] + 0.41 (% G + C)-0.63 (% formamide)-600 / # bp in duplex. As an example of the above formula, [Na ⁺ ] = [0.368] and 50-% formamide, 42%
GC content and average probe size of 200 bases, Tm is 57 ° C. For each 1% decrease in homology, the Tm of the DNA duplex decreases by 1-1.5 ° C. Thus, using a hybridization temperature of 42 ° C., one observes targets with about 75% sequence identity. Such sequences are considered to be substantially homologous to the nucleic acid sequences of the present invention.

It is well known in the art to gradually increase the stringency of hybridization until only a few positive clones remain.
Other suitable conditions are, for example, for detection of sequences having about 80-90% identity.
Includes overnight hybridization at 42 ° C. in 0.25 M Na ₂ HPO ₄ , pH 7.2, 6.5% SDS, 10% dextran sulfate, and a final wash at 55 ° C. in 0.1 × SSC, 0.1% SDS. I do. For the detection of sequences having greater than about 90% identity, suitable conditions include overnight hybridization at 62 ° C. in 0.25 M Na ₂ HPO ₄ , pH 7.2, 6.5% SDS, 10% dextran sulfate. And a final wash at 60 ° C. in 0.1 × SSC, 0.1% SDS.

An alternative that may be particularly suitable when using plant nucleic acid preparations is 5 × SSPE (
Final solution of 0.9 M NaCl, 0.05 M sodium phosphate, 0.005 M EDTA pH 7.7), 5
X Denhardt's solution, 0.5% SDS, overnight at 65 ° C (high stringency, highly similar sequences) or 50 ° C (low stringency, low similarity sequences). 0.2 × SSC / 0.1% SDS at 65 ° C. for high stringency
1 × SSC / at 50-60 ° C. for washing in water or low stringency
Wash in 0.1% SDS. The present invention provides nucleic acids that are capable of selectively hybridizing under high stringency to the nucleic acids identified herein, such as the coding sequence of FIG. 1, the sequence of FIG. 4, or the sequence of FIG. 5C or 5D. Is extended to

PCR techniques for the amplification of nucleic acids are described in US Pat. No. 4,683,195 and Saiki et al., Science
ce 239: 487-491 (1988). PCR involves the steps of denaturing a template nucleic acid (if double-stranded), annealing the primer to a target, and polymerizing. The nucleic acid that is probed or used as a template in the amplification reaction can be genomic DNA, cDNA or RNA. PCR can be used to amplify specific sequences from genomic DNA, specific RNA sequences, and cDNA transcribed from mRNA.

References for general use of the PCR technique include: Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51: 263 (1987); Ehrlich (
Editor), PCR Technology, Stockton Press, NY, 1989; Ehrlich et al., Science
252: 1643-1650 (1991); "PCR protocols: A Guide to Methods and A
pplications ", editor: Innis et al., Academic Press, New York (1990).

Evaluation of whether a PCR product corresponds to a gene capable of altering plant resistance to a pathogen, as discussed, can be performed in a variety of ways, and the PCR band can represent a complex mixture of products. May be contained. Cloning individual products,
Each is then screened for linkage to such known genes that segregates in progeny that exhibit polymorphism for the probe. Alternatively, a PCR product in a manner such that the PCR product can display polymorphisms against a denatured polyacrylamide DNA sequencing gene, having a specific linkage linked to a gene selected before cloning. Can be processed.

Once a candidate PCR bond has been cloned and shown to be linked to a known resistance gene, a clone is isolated that can be tested for other characteristics and homology to Rar1 / Rar1 or other related genes. To this end, PCR binding can be used. It can subsequently be analyzed by transformation to assess its function for the introduction of the plant in question into disease-susceptible varieties. Alternatively, PCR
Sequences derived by binding or analyzing them can be used to assist plant breeders when monitoring the isolation of useful resistance genes.

These techniques are generally applicable to the identification of genes that can alter plant resistance to pathogens. Preferred amino acid sequences suitable for use in designing probes or PCR primers are conserved between at least two Rar1 peptides or polypeptides encoded by genes involved in the signaling of defense responses in plants (complete (Substantially or partially). Conserved sequences can be identified using the information described herein, for example, in FIG.

Based on amino acid sequence information or nucleotide sequence information, oligonucleotide probes or primers can be designed (when studied from amino acid sequence information, the degeneracy of the genetic code and, if appropriate, the biological Consider codon usage).

The gene or fragment thereof identified with which the nucleic acid molecule hybridizes can be an amplified PCR product, which can be isolated and / or purified and subsequently alter the plant resistance to pathogens You can study your ability to If the identified nucleic acid is a fragment of a gene, the fragment can be used in subsequent cloning of the full-length gene (eg, by probing and / or PCR), where the gene can be a full-length coding sequence .

Inserts can be prepared from partial cDNA clones and used to screen cDNA libraries. The activity can be assayed and / or sequenced by subcloning the isolated full-length clone into an expression vector and introducing it into a suitable host cell. It may be necessary to join one or more gene fragments to generate a full-length coding sequence.

A molecule found to manipulate a gene capable of altering plant resistance to infection can be used as such, ie, can be used to alter plant resistance to pathogens. In various aspects of the invention, there are provided nucleic acids obtained and obtainable using the methods disclosed herein.

The present invention also provides an oligonucleotide based on the Rar1 nucleotide sequence provided herein or a Rar1 nucleotide sequence that can be obtained according to the disclosure and suggestions herein. The oligonucleotide may have a suitable length for use as a primer in an amplification reaction, or may be suitable for use as a hybridization fishing probe. Preferably, an oligonucleotide according to the present invention, for example for use in nucleic acid amplification, has about 10 or fewer codons (eg, 6, 7 or 8), ie, about 30 or less (eg, , 18, 21 or 24) nucleotides in length. Probes or primers can be about 20-30 nucleotides in length.

The nucleic acid molecules and vectors according to the invention can be isolated and / or isolated from their natural environment.
Or in purified form, substantially pure or homogeneous, or free or substantially free of nucleic acids and / or genes of origin other than the species in question or the sequence of interest. Can be provided. Nucleic acids according to the present invention include cDNA, RNA, genomic DNA and can be wholly or partially synthetic. The term "isolate" as used herein can encompass any of these possibilities.

[0054] Nucleic acids provided herein or obtainable using the disclosure herein may alter one or more nucleotides (by degeneracy of the genetic code) with or without altering the encoded amino acid sequence. Or it can be the subject of modification by two or more additions, insertions, deletions or substitutions. Such altered forms of the Ral1 nucleotide sequence provided herein or obtainable using the disclosure herein are readily and routinely available for both Ral1 and Ral1 function according to standard techniques. Can be tested. These technologies are basically
Plants or plant cells carrying the mutation, derivative or variant are tested for an altered defense response against the appropriate pathogen.

The nucleic acid molecule can be, for example, in the form of a recombinant, preferably replicable, suitable for use with Agrobacterium, such as a plasmid, cosmid, phage or binary vector. . The nucleic acid can be under the control of a promoter and regulatory elements suitable for expression in a host cell, eg, a microorganism, eg, a bacterium, or a plant cell. genome
In the case of DNA, this can contain its own promoter and regulator, and in the case of cDNA, it can be under the control of appropriate promoters and regulators for expression in a host cell. .

Thus, the nucleotide sequence (for example) of FIG. 1 can be placed under the control of a promoter other than the promoter of the barley Rar1 gene. Similarly, Rar1 homolog sequences from other species can be operably linked to promoters other than the naturally associated promoter. However, vectors containing a nucleic acid according to the present invention need not include a promoter, particularly when the vector is used to introduce a nucleic acid into a cell for recombination into the genome.

The nucleic acids provided by the present invention can be placed under the control of an inducible gene promoter, and thus the expression can be placed under the control of the user. In a further aspect, the invention provides a genetic construct comprising an inducible promoter operably linked to a nucleotide sequence provided by the invention. As discussed, this allows for control of gene expression. The invention also relates to plants transformed with the genetic constructs, and the introduction of such constructs into plant cells and / or the application of, for example, suitable stimulating factors, such as effective endogenous inducers. Thereby providing a method comprising inducing expression of the construct in a plant cell.

The term “inducible” when 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 can be generated intracellularly or provided exogenously. Is done. The nature of the stimulator varies between promoters. Some inducible promoters cause little or undetectable levels of expression (or do not cause expression) in the absence of a suitable stimulus. Other inducible promoters cause detectable constitutive expression in the absence of a stimulator.

[0059] No matter what level of expression is in the absence of the stimulus, expression from any inducible promoter is increased in the presence of the correct stimulus. The preferred situation is
When expression levels are increased by an amount effective to alter the phenotypic properties upon application to the stimulus. Thus, an inducible (or "switchable") promoter that causes a basal level of expression in the absence of a stimulator can be used, the level of which is too low to produce the desired phenotype (and In fact it can be zero). Application of the stimulator increases (or switches on) expression to a level that produces the desired phenotype. One example of an inducible promoter is Cad
Dick et al., (1998) Nature Biotechnology 16: 177-180. Numerous other examples are known to those skilled in the art.

[0060] Other suitable promoters may include the following promoters: the promoter of the cauliflower mosaic virus 35S (CaMV 35S) gene, which is expressed at high levels in virtually all plant tissues (Benfey et al. , (1990) EMBO J.
9: 1677-1684); the cauliflower meri 5 promoter, which grows in apical meristems and in some well-localized locations in plants, such as the medial phloem, flower primordia, roots and shoots Expressed at the branch point (Medford, J
.I. (1992) Plant Cell 4: 1029-1039; Medford et al., (1991) Plant Cell 3
: 359-370) and Arabidopsis thaliana LEAFY.
Promoter, which is expressed very early in flower development (Weigel et al., (19
92) Cell 69: 843-859).

One of skill in the art is well able to construct vectors and design protocols for expression of recombinant genes. A suitable vector can be selected or constructed containing appropriate regulatory sequences, such as a promoter sequence, terminator fragment, polyadenylation sequence, enhancer sequence, marker gene and, where appropriate, other sequences. For further details, see the following documents: Molecular Cloning: A Laboratory Manual: Second Edition, Sambrook
1989, Cold Spring Harbor Laboratory Press.

For example, in the manufacture of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and expression of genes, and analysis of proteins, a number of known techniques and protocols for manipulating nucleic acids are described below. Described in detail in: Curr
ent Protocols in Molecular Biology, 2nd edition, Ausubel et al., Ed., John Wile
y & Sons, 1992. The disclosures of Sambrook et al. And Ausubel et al. Are hereby incorporated by reference.

Certain procedures and vectors that have been widely and successfully used conventionally for plants include:
It is described in the following literature: Bevan, Nucl. Acids Res. 12: 8711-8721 (1
984) and Guerineau and Mullineaux (1993) Plant transformation and
expression vectors. In: Plant Molecular Biology Labfax (Croy RRD e
d) Oxford, BIOS Scientific Publishers, pp. 121-148.

[0064] Selectable genetic markers can be used. Such markers are
Consists of a chimeric gene that confers a selectable phenotype, eg, resistance to antibiotics, eg, kanamycin, hygromycin, phosphinothricin, chlorsulfuron, methotrexate, gentamicin, spectinomycin, imidazolinone, and glyphosate.

When introducing the selected gene construct into the cell, certain considerations well known to those skilled in the art must be considered. The nucleic acid to be inserted should be assembled in a construct containing effective regulatory elements that drive transcription. Methods for transporting the construct into the cell are available. Once the construct is located within the cell membrane, integration into endogenous chromosomal material will or will not occur. Finally, as far as plants are concerned, the type of target cell must be such that cells can be regenerated in the whole plant.

Plants transformed with a DNA segment containing the sequence can be produced by standard techniques already known for genetic manipulation of plants. The DNA can be transformed into plant cells using any suitable technique, for example, the following technique: Agrobacterium, which takes advantage of its natural gene transfer capabilities.
erium) carrying a disarmed Ti-plasmid vector (EP-A-27
0355, EP-A-0116718, NAR 12 (22) 8711-87215 1984), particle or microprojectile impact (US 5100792, EP-A-444882, EP-A-434616),

Microinjection (WO 92/09696, WO 94/00583, EP 331083, EP
175966, Green et al. (1987) Plant Tissue and Cell Culture, Academic Pr
ess), electroporation (EP 290395, WO 8706614), other forms of direct DNA absorption (DE 4005152, WO 9012096, US 4684611), liposome-mediated DNA absorption (eg, Freeman et al., Plant Cell Physiol. 29: 1353). (1984), or a vortex formation method (eg, Kindle, PNAS USA 87: 1228 (1990d). The physical method of transformation of plant cells is described in Oard, 1991, Biotech. Adv. 9: 1-11.

Thus, once a gene has been identified, the gene is reintroduced into plant cells using techniques well known in the art to produce a transgenic plant of the appropriate phenotype. can do. In order to transform dicotyledonous species in this field, Agrobacterium (
Transformation of Agrobacterium) is widely used. The production of stable, fertile transgenic plants in almost all economically relevant monocotyledons is also routine:

(Toriyama et al. (1998) Bio / Technology 6: 1072-1074; Zhang et al. (1988) Pla.
nt 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 / Tec.
hnology 8: 736-740; Christou et al. (1991) Bio / Technology 9: 957-962; Pe
ng et al. (1991) International Rice Research Institute, Manila, Philippine
s 563-574; Cao et al. (1992) Plant Cell Rep. 11: 585-591; Li et al. (1993) P
lant Cell Rep. 12: 250-255; Rathore et al. (1993) Plant Molecular Biology.
21: 871-884;

Fromm et al. (1990) Bio / Technology 8: 833-839; Gordon-Kamm et al. (1990) Pla.
nt Cell 2: 603-618; D'Halluin et al. (1992) Plant Cell 4: 145-1505; Wa
(1992) Plant Molecular Biology 18: 189-200; Koziel et al. (1993) Bi.
otechnology 11: 194-200; Vasil, IK (1994) Plant Molecular Biology 2
5: 925-937; Weeks et al. (1993) Plant Physiology 102: 1077-1084; Somers et al. (1992) Bio / Technology 10: 1589-1594; WO 92/14828). In particular, Agrobacterium-mediated transformation is currently another highly efficient transformation method in monocotyledonous plants (Hiei et al. (1994) The Plant Journal 6:27).
1-282).

The emergence of fertile transgenic plants occurs in cereals, rice, corn, wheat,
Achieved in oats and barley (reviewed in the following literature: Shimamoto, K. (1994) Current Opinion in Biotechnology 5,158.
-162; Vasil et al. (1992) Bio / Technology 10: 667-674; Vain et al., 1995, Biote.
chnology Advances 13 (4): 653-671; Vasil, 1996, Nature Biotechnolog
y 14 p.702). Wan and Lemaux (1994) Plant Physiol. 104: 37-48
Techniques for generating a large number of independently transformed fertile barley plants are described.

When Agrobacterium is inadequate or ineffective, microprojectile bombardment, electroporation and direct DNA absorption are preferred. Alternatively, using a combination of different techniques, the transformation process, for example, impact using Agrobacterium-coated microparticles to induce wounds (EP-A
-486234) or enhance the efficiency of microprojectile bombardment, followed by Agrobacterium (
Agrobacterium) (EP-A-486233).

After transformation, the plants can be transformed, for example, into single cells, as is standard in the art,
It can be regenerated from callus tissue or leaf disks. Almost any plant can be completely regenerated from plant cells, tissues and organs. Available techniques are reviewed in the following literature: Vasil et al., Cell Culture and Somat.
ic Cell Genetics of Plants, Vol. I, II and III, Laboratory Proced
ures and Their Applications, Academic Press, 1984, and Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press.
, 1989.

The particular choice of transformation technique will be dictated by its efficiency in transforming a plant species and the experience and preferences of those practicing the invention according to the particular method chosen. It will be appreciated by those skilled in the art that the particular choice of transformation system to introduce the nucleic acid into the plant cells, as well as the choice of techniques for plant regeneration, is not essential or is not a limitation of the present invention. Will be obvious to Furthermore, the invention encompasses host cells, especially plant or microbial cells, transformed with the aforementioned vectors. Thus, a host cell, eg, a plant cell, comprising the nucleic acid sequences provided herein is provided. In a cell, the nucleotide sequence can be integrated into the chromosome.

Further, according to the present invention, there is provided a plant cell having a nucleotide sequence, particularly a heterologous nucleotide sequence, provided in the genome thereof, provided by the present invention under the operative control of a regulatory sequence for controlling expression. Provided. A coding sequence can be operably linked to one or more regulatory sequences. A regulatory sequence may be heterologous or foreign to the gene, for example, it may not naturally associate with the gene for its expression. The nucleotide sequence according to the invention can be placed under the control of an externally inducible gene promoter, and the expression can be placed under the control of the user.

Another aspect of the present invention provides a method for producing such a plant cell. This method involves introducing a nucleotide sequence or a suitable vector containing a nucleotide sequence into a plant cell, causing recombination between the vector and the plant cell genome, or possibly introducing the nucleotide sequence into the genome. It is included. The present invention extends to plant cells containing a nucleotide sequence according to the present invention as a result of introducing the nucleotide sequence into an ancestral cell.

The term “heterologous” is used to indicate that the gene / nucleotide sequence in question has been introduced into said plant cell or its ancestry using genetic engineering, ie, due to human involvement. Used for Transgenic plant cells can be provided, ie, plant cells that are transgenic for the nucleotide sequence in question. The transgene can be present on an extra genomic vector or, preferably, stably integrated into the genome. A heterologous gene can replace an endogenous equivalent gene, ie, a gene that normally performs the same or similar function, or the inserted sequence can be additional to the endogenous gene or other sequence.

An advantage of heterologous gene transfer is that the expression of a sequence can be placed under the control of a selected primer to affect expression as desired. Further, for example,
Wild type mutations, variants and derivatives having higher or lower activity than wild type can be used in place of the endogenous gene. A nucleotide sequence that is heterologous or exogenous or foreign to the plant cell can be one that is not naturally present in its type, variant or species of cell. Thus, a nucleotide sequence can include a coding sequence of or derived from a particular type of plant cell or plant species or varieties, arranged within the context of different types or species or varieties of plants. .

One further possibility is to place the nucleotide sequence in a cell where the nucleotide sequence or homolog is naturally found, however, wherein the nucleotide sequence is a plant cell, or One or more regulatory sequences linked to and / or present adjacent to a nucleic acid that is not naturally present in a plurality of cells or species or variants of that type, for example, for control of expression,
For example, it is operably linked to a promoter sequence. Sequences in plants or other host cells can be identifiably heterologous, exogenous or foreign.

A plant comprising a plant cell according to the invention is also provided with any part or ovule, seed, inbred or hybrid progeny or progeny thereof. In particular, there is provided a transgenic crop plant that has been engineered to carry a gene identified as described above. Examples of suitable plants include tobacco, cucumber, carrot, vegetable brassica, pepper, grape, lettuce, strawberry, amazuna, sugar beet, wheat, barley, corn, rice, soybean, pea, sorghum, sunflower, tomato,
Includes potato, pepper, chrysanthemum, carnation, poplar, eucalyptus and pine.

A plant according to the invention can be a plant that does not truly breed in one or more properties. Plant breeders 'rights, especially plant breeders' rights
), Plant varieties that can be registered can be excluded. Since the plant stably contains in its genome a transgene introduced into its cells or its ancestors,
It is recognized that plants are not necessarily considered simply “plant varieties”.

In addition to plants, the present invention provides such plants, seeds, progeny or hybrid progeny or progeny clones, and any part thereof, such as cuttings, seeds. The present invention provides osteophytes of any plant, i.e., sexual or asexual, any part that can be used in reproduction or propagation, such as cuttings, seeds and the like. Also, sexually or asexually progeny progeny of such plants, plants that are clones or progeny, or any parts or ovules, progeny, clones or progeny of said plants are within the scope of the invention.

The present invention also includes the peptide expression products of the nucleic acid molecules according to the present invention, which can be disclosed herein or obtained according to the information and suggestions herein.
Also provided is a method for producing such an expression product by expression from a nucleotide sequence encoding it under suitable control in a suitable host cell, eg, E. coli. One of skill in the art is well capable of constructing vectors and designing protocols and systems to express and recover the products of recombinant gene expression.

Preferred polypeptides include the amino acid sequence shown in FIG. Peptides according to the present invention may be alleles, variants, fragments,
It can be a derivative, mutation or homolog. The allele, variant, fragment, derivative, mutation or homologue is the amino acid sequence Rar1 shown in FIG.
It can have substantially homologs or be a Rar1 mutation.

[0085] Also clearly related to functional Rar1 polypeptides (eg, they are immunologically cross-reactive with Rar1 polypeptides exhibiting Rar1 function, or characteristic sequence motifs common to Rar1 polypeptides) Polypeptides which no longer have Rar1 function are encompassed by the present invention. Thus, the invention provides variants of the Rar1 polypeptide, eg, variants resulting from the rar1-1 and rar1-2 mutations identified herein. Plants and plant cells carrying these mutants are:
Sensitive to pathogen invasion.

“Homology” with respect to amino acid sequence is used to refer to identity or similarity, preferably identity. As already mentioned above, high levels of amino acid identity are restricted to functionally significant domains or regions, such as any of the domains identified herein (see, eg, FIG. 6).

In particular, homologs of the specific Rar1 polypeptide sequences provided herein, as well as mutations, variants, fragments and derivatives of such homologs are provided by the present invention. Such homologs can be readily obtained by using the disclosure made herein. Thus, the invention also extends to polypeptides that can be obtained using the sequence information provided herein.

A Rar1 homolog has homology to the amino acid sequence of FIG. 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%, or at least about 85%, or at least about 88%, or at least about 90% homology, most preferably at least about 95% or higher homology At the amino acid level.

In certain embodiments, an allele, variant, derivative, mutant derivative, mutation or homolog of a particular sequence has a slight overall homology to the particular sequence, eg, about 20%, or About 30%, or about 35%, or about 40%, or about 45% homology may be exhibited. However, in functionally significant domains or regions, amino acid homology can be very high.

[0090] Putative functionally significant domains or regions can be identified using bioinformatics processes, including comparison of homologous sequence sequences. Functionally significant domains or regions of different polypeptides can be combined for expression as a fusion protein from the encoding nucleic acid. For example, the advantageous or desirable properties of different homologs can be combined in a hybrid protein such that the resulting expression product having Rar1 or Rar1 function can include fragments of various parent proteins.

The individual domains and fragments of the Rar1 polypeptide are shown in FIG. 6, and as described above, these, and also derivatives, variants and homologs, in various aspects and embodiments of the present invention. For example, useful in activating cell death and / or downstream resistance responses.

Amino acid sequence similarities are as defined and can be determined by the TBLASTN program described in Altschul et al. (1990) J. M.
ol. Biol. 215: 403-10, which is a standard use in this field. In particular,
TBLASTN 2.0 can be used with Matrix BLOSUM62 and GAP penalties: presence: 11, extension: 1. Other standard programs that can be used are B
estFit, which is Wisconsin Package, version 8, September 1994 (Genetics
Computer Group, 575 Science Drive, Madison, Wisconsin, USA, Wiscons
in 57311). BestFit creates an optimal alignment of the best segment of similarity between two sequences.

Optimal alignment by inserting gaps to maximize the number of matches using the local homology algorithm of Smith and Waterman (Adv. Appl. Math. (1981) 2: 482-489). Is found. Other algorithms include a GAP that increases the number of matches and minimizes the number of gaps, which neat to align two complete sequences.
Uses the edleman and Wunsch algorithms. Whichever algorithm is used, the default parameters are generally used, these are the gap penalty for GAP = 12 and gap extension = 4. Alternatively, a gap penalty of 3 and a gap extension of 0.1
Can be used. The algorithm FASTA (this is the Pearson and Lipman (19
88) The method of PNAS USA 85: 2444-2448) is a further alternative.

The use of both the terms “homology” and “homologous” herein means, for example, that “homology” simply requires that the two sequences have sufficient similarity to recombine under the appropriate conditions. Consistent with terms such as "target recombination," it does not imply the necessary evolutionary relationship between the compared sequences. The polypeptide according to the invention, which can be encoded by the nucleic acid according to the invention, is further described below.

In accordance with techniques standard in the art, purified Rarl polypeptides and their mutations, variants, fragments, derivatives, produced by expression from encoding nucleic acids, for example, to generate antibodies, are provided. Alleles and homologs can be used. As further described below, polypeptides, including antibodies and antigen-binding fragments of antibodies, can be used, particularly when identifying homologues of the sequences provided herein.

[0096] Methods of producing antibodies include immunizing a mammal (eg, human, mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or fragment thereof. Antibodies can be obtained from immunized animals using a variety of techniques known in the art, and can be screened, preferably using the binding of the antibody to the antigen in question. For example, Western blot techniques or immunoprecipitation can be used (Armitage et al., 1992, Nature 35
7: 80-82).

As an alternative or an adjunct to immunization of mammals, expressed, for example, using lambda bacteriophage or filamentous bacteriophage displaying functional immunoglobulin binding domains on their surface Antibodies with suitable binding specificities can be obtained from recombinantly produced libraries of immunoglobulin variable domains, see, for example, WO 92/01047.

An antibody raised against a polypeptide or peptide can be obtained by homologous polypeptide,
It can then be used in the identification and / or isolation of the coding gene. Thus, the present invention provides polypeptides having Rar1 function or methods of identifying or isolating Rar1 function (according to embodiments disclosed herein). The method involves screening a candidate polypeptide or polypeptide with a polypeptide comprising the antigen binding domain of an antibody (whole antibody or fragment thereof), wherein the antibody is a Rar1 or a Rar1 peptide, polypeptide or They can bind to their fragments, variants or derivatives, or preferably have binding specificity for such a peptide or polypeptide, eg, those having the amino acid sequences identified herein.

A specific binding member comprising an antigen binding domain, which binds to, or is preferably specific for, Rar1 or a peptide, polypeptide or mutation, variant or derivative thereof, of Rar1; For example, antibodies and polypeptides, and their uses and methods of using them, represent another aspect of the invention. A candidate peptide or polypeptide for screening is, for example, the product of an expression library created using nucleic acids from the plant in question,
Alternatively, it can be the product of a purification process from a natural source.

A peptide or polypeptide found to bind to an antibody can be isolated and then subjected to amino acid screening. The peptide or polypeptide can be completely or partially sequenced using any suitable technique (eg, fragments of the polypeptide can be sequenced). When obtaining a nucleic acid encoding a peptide or polypeptide, information on the amino acid sequence can be used. For example, one or more oligonucleotides (eg, a degenerate pool of oligonucleotides) are designed for use as probes or primers in hybridization to a candidate nucleic acid, or a computer sequence database is searched. These will be described later.

The present invention further provides a method of promoting cell death and / or a plant pathogen defense response in a plant. The method involves expressing a heterologous nucleic acid sequence having the aforementioned Rar1 function in a plant cell. The present invention further provides a method for generating pathogen resistance in a plant, comprising expressing a heterologous nucleic acid sequence having the aforementioned Rar1 function in a plant cell.

Such a method can be achieved as follows. After performing the initial steps of introducing the nucleotide sequence into the plant cell or its ancestors, expression from the nucleotide sequence encoding the amino acid sequence conferring Rar1 function is performed in the plant cell (whereby the encoded poly Produce peptides). Such a method can generate plant resistance to pathogens.

The expression of Rar1 transcript or Rar1 protein can be manipulated to enhance resistance to a broad spectrum of pathogens in different plants. This can be achieved by overexpression using a highly active plant promoter, for example, the CaMV-35S promoter. Alternatively, Rar1 is linked to a pathogen-inducible promoter (see description below) to increase expression in challenged cells. Increased disease resistance can occur in the absence of a hypersensitivity response (HR), which can have deleterious effects on plants with respect to general vitality and yield.

The gene stably integrated into the plant genome is transmitted from generation to generation to the progeny of the plant, and the progeny cells are capable of expressing the encoded polypeptide;
Thus, it may have enhanced pathogen resistance or pathogen sensitivity. By assessing the suitability of the pathogen as described above, pathogen resistance can be determined.

The invention further provides that after performing the initial steps of introducing the nucleic acid into a plant cell or its ancestor, the mutation, allele or derivative of the amino acid sequence of FIG. The present invention provides a method comprising performing expression from a nucleotide sequence encoding (which can have function) in a plant cell, thereby producing the encoded polypeptide. Such a method can generate plant resistance to one or more pathogens. This method is described in WO 91/1558
5 (Mogen), more preferably with the avr gene according to any of the methods described in PCT / GB95 / 01075 (published as WO 95/31564) or any other involved in conferring pathogen resistance Can be used in combination with the gene.

In the present invention, resistance can be altered by introducing a nucleotide sequence in the sense direction. Thus, the present invention provides a method of modulating a defense response in a plant. The method involves causing or enabling expression of the nucleic acid in a plant cell according to the present invention. Generally, it will be desirable to promote a protective response. This can be achieved by allowing the Rar1 gene to function. To down regulate the resistance signaled by Rar1, expression of the endogenous Rar1 gene can be reduced using antisense technology or "sense modulation".

It has now been established to use antisense genes or partial gene sequences to down regulate gene expression. Double-stranded DNA is placed "in the reverse direction" under the control of a promoter, and thus transcription of the "antisense" strand of DNA
Complementary to normal mRNA transcribed from "sense" strand of target gene
Allow RNA to occur. The complementary antisense RNA sequence then binds to the mRNA to form a duplex, which would inhibit the translation of endogenous mRNA from the target gene into protein. It is not yet clear whether this is the actual mode of operation.

However, it is an established fact that this technique is effective. See, for example, the following references: Rothstein et al., 1987; Smith et al. (1988) Nature 334.
: 724-726; Zhang et al. (1992) The Plant Cell 4: 1575-1588; English et al. (1
996) The Plant Cell 8: 179-188. In addition, antisense technology, Bourque, 1
995, and Flavell, 1994. The antisense construct is Ra
It may include the 3 'or 5' end of the r1 or Rar1 homolog. Some Rar1
If the homolog is present in a plant species, the inclusion of 5 ′ and 3 ′ untranslated sequences in the antisense construct will enhance silencing specificity.

Using a native promoter, a constitutively expressed promoter, such as Ca
The construct can be expressed by the MV 35S promoter, by a tissue-specific or cell-type-specific promoter, or by a promoter that can be activated by external signals or factors. CaMV 35S promoter, but also rice actin1
And corn ubiquitin promoters have been shown to express reporter genes at high levels in rice (Fujimoto et al. (1993) Bio / Technol).
ogy 11: 1151-1155; Zhang et al. (1991) Plant Cell 3: 1155-1165; Cornejo
(1993) Plant Molecular Biology 23: 567-581).

The complete sequence corresponding to the coding sequence in the opposite direction need not be used. For example, fragments of sufficient length can be used. It is routine for those skilled in the art to screen fragments of different sizes and optimize antisense inhibition from different parts of the coding sequence. It may be advantageous to include ni1 or more nucleotides upstream from the initiation methionine ATG codon and possibly the initial codon. Suitable fragments are about 14 to 23 nucleotides, e.g., about 15
, 16 or 17 nucleotides.

Thus, the present invention provides a method of down-modulating Rar1 expression in plants. This method involves causing or enabling antisense transcription from a nucleic acid in a plant cell according to the present invention. Down-regulation of Rar1 can reduce defense responses. This is in some circumstances, for example,
It may be appropriate as an analytical or experimental approach.

Complementary to the coding sequence of the Rar1 gene (ie, including homologues) for use in antisense regulation, or a fragment of said coding sequence suitable for use in antisense regulating expression. Provided are nucleic acids that include a nucleotide sequence of interest. It is DNA and can be under the control of regulatory sequences appropriate for antisense transcription in the cell in question.

When an additional copy of the target gene is inserted in the sense, ie, in the same orientation as the target gene, a range of phenotypes is produced, and these phenotypes can be Includes some individuals where low expression of the protein occurs. When the inserted gene is only a part of the endogenous gene, the number of low expressing individuals in the transgenic population increases.

The mechanism by which sense regulation occurs, particularly down regulation (or “silencing”), is not well understood. However, this technique is also well documented in the scientific and patent literature and is used routinely for gene regulation. See, for example, the following: van der Krol et al. (1990) The Plant
Cell 2: 291-299; Napoli et al. (1990) The Plant Cell 2: 279-289; Zhan
g et al. (1992) The Plant Cell 4: 1575-1588. Further improvements in gene silencing or co-suppression techniques have been described in WO 95/34668 (B
iosource); Angell and Baulcombe (1997) The EMBO Journal 16, 12: 367
5-3684; and Vonnet and Baulcombe (1997) Nature 389: p. 553.

Suitable fragments are about 50, 100, 150, 200, 250, 300, 350, 400, 450, 50
It can be 0, 550, 600, 650 or 700 nucleotides in length. Thus, the present invention also provides a method of down-modulating Rar1 function in plants. This method involves causing expression from a nucleic acid in a plant cell or, if possible, suppressing endogenous Rar1 expression in accordance with the present invention. Using a modified version of Rar1, endogenous Rar1 function can be down-regulated. For example, mutations, variants, derivatives, and the like can be used.

The wild-type activity of Rar1 can be reduced by using a ribozyme, eg, replication replication, eg, a hammerhead class ribozyme (Hase
loff and Gerlach, 1988, Nature 334: 585-591; Feyter et al., 1996, Mol. Ge.
n. Genet. 250: 329-338).

In another method for reducing Rar1 function in plants, use transposon mutagenesis (Osborne et al. (1995) Current Opinion in Cell Biology).
7: 406-413). Gene inactivation has been demonstrated using an endogenous mobile factor or a heterologous cloned transposon that retains mobility in the foreign genome via a "targeted labeling" approach. By using PCR primers with binding specificity in both the inserted sequence and the Rar1 homolog, one can identify Rar1 alleles carrying insertions of known sequence.
To stabilize the transposon in inactivated cells, a "two-factor system" can be used.

In a two-factor approach, a T-DNA is constructed that supports a non-autonomous transposon containing a selectable or screenable internal marker gene inserted among excision markers. A plant that supports the second T-DNA that expresses the transposon function is crossed with a plant that supports these T-DNAs. Select double for markers in excision and transposon hybrid to produce F ₂ plants with the transferred factors.

With reference to the drawings, embodiments and examples relating to the present invention will now be described. All documents cited herein are hereby incorporated by reference.

Embodiment 1 Cloning and cloning of Rar1 from barley and homologues from other species
The interval mapping procedure genetic mapping previous low resolution high resolution of fine characterization RAR1, RAR1 is flanked by RFLP loci cMWG694 and MWG503 in 5cM interval was found on barley chromosome 2 (Freialdenhoven Et al., 1994). 1cM in barley corresponds to almost 3Mb,
As a first step in isolating Rar1, we decided to establish a local, high-resolution genetic map. We aimed for a resolution of approximately 0.01 cM, corresponding to an average physical distance of 30 kb.

The efficacy of intercrossing is advantageous because it increases the likelihood of finding a polymorphism with a given probe, especially due to the low frequency of DNA polymorphism in the spring form (Russel et al., 1997) , We justified. Therefore, both mutations were transformed into three Mla-12 backcross (BC) lines (Mla-12 BC Ingrid, Mla-12 BC Pallas, Mla-12 BC Siri
(Freialdenhoven et al., 1994; Kolster et al., 1986; Kolster and Stolster 1987).
). These lines represent a different genetic background of the barley spring type.

The steps of high-resolution mapping were, in summary, as follows: Using an initial RFLP map based on 50 plants (Freialdenhoven et al., 1994), CAPS
Markers MWG876, MWG892 and MWG2123 were incorporated into the genetic map. Ant2 and Rar1
A phenotypic screen was used to analyze 1040 plants for recombination events between and. Using the observed recombinants, the MWG892 melting point was determined to be distal with respect to Rar1. At the marker interval MWG892-cMWG694, a subsequent CAPS-based recombination screen of 1063 additional plants was performed. Analysis of the observed recombinants localized MWG876 proximal to Rar1.

Finally, another 2207 plants were studied in the marker interval MWG892-MWG876.
Two AFLPs R25 / 48 and A25 / 43 were identified and tested for DNA from individuals in the pool. R25 / 48 was identified in the resistant population segregants. P
The CR product was analyzed on a 4.5% denaturing polyacrylamide gel. The presence of a linked AFLP signal was found in one of the individuals in the pool, indicating a recombination event between the AFLP loci R25 / 48 and Rar1, which was not displayed in the sensitive pool (Bs). Two recombinants identified using R25 / 48 and A25 / 43 reveal co-segregation of Rar1 and MWG876, and thus both AFLP loci must be located distal to MWG876. Genetic distance (cM) was calculated based on two-point estimates.

In more detail: Approximately 5 cM containing Rar1 was converted to RFLP markers MWG503 and cMWG694 (Freialdenhoven
Et al., 1994), oligonucleotides for amplification of the corresponding loci were derived, and susceptible parents (rar1-1, rar1-2) and resistant parents (Mla-12 BC Ing).
rid, Mla-12 BC Pallas, Mla-12 BC Siri).

This is because M82, M100, Mla-12 BC Ingrid, and Mla-12 BC Pall were used as template DNA.
Displaying the restriction digested amplification products on ethidium bromide stained 2.5% agarose gels using as and Mla-12 BC Siri. Amplification and digestion were performed as described below with respect to Table 2. The displayed CAPS markers corresponded to the RFLP loci MWG87, MWG503, MWG876, and MWG2123. PCR primers for locus MWG892 allowed allele-specific discrimination of PCR products without subsequent restriction digestion. The small band was due to an incomplete BclI digest of the PCR amplicon.

To increase the marker density adjacent to Rar1, the general RFLP map
We have selected three more RFLP markers that map closely to the LP locus (MWG876, MWG892 and MWG2123 [Graner, 1991]. An amplifiable polymorphism capable of cleaving each of these RFLPs. Sequence (CAPS) and mapped for Rar1 based on a population of 50 segregants, in which MWG876 and MWG8
92 showed cosegregation with Rar1, while MWG2123 was located distal to cMWG694.

Recombinant screening Cultivar Sltan-5 (Rar1-1 and rar1-2) that induced both Rar1 mutations (rar1-1 and rar1-2)
Mla-12, Rar1) contains an anthocyanin pigmentation defect (ant2), but the three resistant Mla-12 BC lines used for mapping (Mla-12 BC Ingrid, Mla-12 BC)
Pallas, Mla-12 BC Siri) carries the Ant2 wild-type allele. The Ant2 locus has been shown to map proximally to Rar1 at a distance of approximately 0.5 cM (Freialdenhoven et al., 1994). To identify rare recombinants in smaller spacing between the Rar1 and Ant2, we select the 1,040 susceptible F ₂ individuals (rar1 / rar1),
Screened for the presence of the anthocyanin wild-type allele Ant2.

A total of 14 recombinants were found, which were tested for alleles in MWG87, MWG876 and MWG892. Analysis of the 14 recombinants revealed that Rar1, MWG87 and M
Co-segregation of WG876 was shown. Marker MWG892 was located between Ant2 and Rar1 and was separated from the former by two recombination events. Two allele Rar1 mutations and 3
Different crosses between the two Mla-12 BC lines did not reveal significantly different recombination frequencies. Therefore, we limited our search to only one cross (M10
0 × Mla-12 BC Ingrid). This also allowed the use of recombinants identified adjacent to Rar1 for the targeted AFLP-based screening described below.

[0129] To increase the genetic resolution in the vicinity of the target locus, by utilizing the CAPS markers MWG892 and CMWG694, the recombination event flanking the RAR1, were further screened F ₂ plants 1,063. MWG87 and
Subsequent studies of MWG876 revealed complete linkage for MWG87 and Rar1, except for one recombination event between MWG876 and the target gene, and this RFLP
Was located distal to Rar1.

[0130] Further markers intervals MWG876 by CAPS analysis of F ₂ plants 2,207 - by testing for recombination events in MWG892, were we attempted to genetically isolate the MWG87 from RAR1. However, studies of the observed recombinant plants further revealed co-segregation of Rar1 and MWG87. MWG87, MWG876 and Rar1
Can be indicative of a small physical distance between these loci, but may also be the result of a low recombination frequency in this genetic segment.

To investigate these possibilities and enrich the interval using additional DNA markers, we have begun searching for AFLP-based markers. If the close genetic linkage of Rar1, MWG87 and MWG876 is caused by suppression of recombination, large physical spacing would be expected to reveal multiple linked AFLP markers in the target region.

[0132] By using the selected F ₂ progeny of the targeted AFLP search hybridization rar1-2 × Mla-12 BC Ingrid markers, populations of segregant analysis (Giovannoni, 1991; Michelmore et al., 1991) in combination with, AFLP Technology (Vos et al., 1995) was used. To minimize the detection of AFLP markers that are not tightly linked to Rar1, we used DNA marker-selected recombinants to construct the DNA pool we identified in the aforementioned CAPS-based recombination screen. .

[0133] Both DNA pool was comprise a respective 10 F ₂ plants. When starting the AFLP marker screening analyzes only F ₂ individuals 500 in the marker interval MWG892-cMWG694, recombinants between MWG876 and Rar1 was not yet identified. Eventually, MWG876 could not be used to select appropriate recombinants. The susceptibility pool (rar1-2 / rar1-2) contains three individuals with recombination between cMWG694 and Rar1,
It contained four individuals with recombination between MWG892 and Rar1, and three susceptible individuals without recombination at the marker interval studied.

The selection of recombinants for the resistance pool was based on DNA markers only. cMWG694
By using plants that exhibited an allelic pattern of resistant parents of MWG892 and MWG892, homozygosity could be guaranteed at the corresponding genetic intervals. Thus, linked AFLP markers are expected to be trans and cis. Plants carrying recombination events between MWG503 and MWG892 (two plants) or between cMWG694 and MWG2123 (two plants) were used to narrow the target spacing of the resistance pool. In addition to the recombinant individuals, plants without detectable recombination events at the marker interval studied were used.

The frequency of the genome width of the AFLP polymorphism between M100 and Mla-12 BC Ingrid was found to be 7%. Each AFLP primer combination displayed an average of 100 DNA fragments. Therefore, approximately 40,000 loci were examined using 7 PstI + 2 primers and 56 MseI + 3 primers in 392 combinations. The combination of only two primers identified an AFLP marker linked to Rar1 in the DNA pool. When analyzing these AFLP markers on individuals in each pool, they were separated by one (R25 / 45) and two (A25 / 46) recombination events from the target gene and mapped distally with respect to Rar1. You.

The small amount of the identified Rar1-linked AFLP marker is certainly affected by the way we assemble the DNA pool. Also, small genetic intervals searched for DNA markers can indicate that they are not physically too large. To more accurately estimate the relationship between genetic and physical distances, rare cutting (rare
cutting) PFGE Southern analysis was performed in combination with restriction enzymes and Rar1 linked RFLP probe.

Extensive physical mapping The co-separable probe MWG87 and flanking probes MWG892 and MWG876 were used to determine the size of the fragment after restriction with seven different rear cutting restriction endonucleases. The analysis revealed a single co-migrating MluI restriction fragment that hybridized to MWG87 and MWG876 (Table 1).
). This can indicate a maximum physical distance of 550 kb between MWG876 and MWG87.
Medium size fragments were also detected using the probe / restriction enzyme combinations MWG876 / NotI, SalI and SmaI (90 kb) and MWG87 / SfiI and SmaI (100 kb). These fragments of normal size using one probe and different endonucleases are probably caused by the previously reported grouping of restriction sites in vertebrates (Bickmore et al., 1992; Larsen et al., 1992).

Physical Limitation of Rar1 on Barley Yeast Artificial Chromosomes (YACs) Screened for the barley YAC library bounded by the loci MWG892 and MWG876 that encompass the target locus.
Identification of the 0.7 cM interval was significant in the high resolution genetic mapping of Rar1. Based on more than 8,000 meiosis, locus MWG87 was found to co-segregate with the target. This provided a rationale for initiating the screening of the barley YAC library to isolate a large insert genomic clone containing MWG87.

Performing a PCR-based screen using the truncated and amplified polymorphic sequence (CAPS) MWG87 identified five yeast clones, each generating the expected PCR product. To confirm that the resulting amplicon displayed the MWG87 locus, each PCR product was sequenced and found to be identical in all YAC-derived fragments. Two of the five YACs also contained the CAPS marker MWG876, which maps 0.015 centimorgan (cM) distal to Rar1.

Analysis of YAC Inserts by PFGE and Inverse PCR (IPCR) To determine the insert size of isolated YACs, probe MWG87 was used in PFGE Southern analysis; 680 kb (Y18), 340 kb (Y30), respectively. 1,100kb (
Y31), 720kb (Y73) and 300kb (Y113). In two independent Southern analyses, Y
The signal intensity for AC Y113 was shown to be significantly reduced, demonstrating that the inheritance of this yeast artificial chromosome was disrupted. To establish local contigs using the identified YACs, the left (L) and right (R) ends of the YACs were isolated by IPCR and their nucleotide sequences determined.

Analysis of their YAC-terminal sequence revealed that the YAC-terminal Y31L contained about 95% of the barley BARE-1 retrotransposon, a highly repetitive sequence comprising about 6.7% of the barley genome. It has been shown to have identity (Suoniemi et al., 1996). In addition, sequence stretches with a high relationship to the BARE-1 retrotransposon were found in Y18R, Y18L, and Y73L. In addition, YAC and Y
31R revealed approximately 64% sequence identity to the maize retrotransposon Opie-2 (SanMiguel et al., 1996), indicating a novel factor not previously described in barley.

Overlap analysis of YAC inserts Each probe from each yeast clone was tested against all YACs to determine their relationship. Based on nucleotide blast results, duplicate non-repetitive DNA
A YAC end-specific oligonucleotide was designed in the sequence stretch representing This was not possible for the YAC-terminal Y31L, which comprised only multicopy DNA. Subsequently, the presence or absence of these sequences in each yeast clone was determined using PCR analysis (Table 2). Note that two uncharacterized YACs (Y1, Y2) were included to expose primer pairs that recognize the repetitive DNA.

The amplification product corresponding to the YAC terminus Y31L in yeast clones Y1 and Y2 confirmed the multicopy properties of this YAC terminus. YAC terminal Y113L generated amplification products in YAC Y73 and Y113. However, the length of the PCR product in Y73 was different from the expected size 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 a clear absence / presence on different YAC clones and did not amplify the fragment in Y1 or Y2, indicating that it was suitable for analysis of YAC contigs .

If all YAC inserts are co-linear with the source DNA, each end probe will detect the yeast clone from which it is derived and the YAC covering this region.
For the two YAC ends that make up the ends of the contig, the yeast clone from which they are derived will be detected. Here, these end probes define the ends of the YAC contig. At least two of the four YAC ends must be derived from chimeric YAC, since four YAC ends that are not amplified in YAC have been determined, except for the one from which they were derived. However, based on this information, it remained uncertain that two of the four YAs were chimeric. Genetic mapping of the YAC terminus could resolve this lack of clarity, but is only possible if the terminal probe is a single or low copy marker.

Except for the YAC-terminal copy number Y31L, all YAC-end-specific markers proved to be suitable for establishing YAC DNA-based contigs. Next, it was determined whether the oligonucleotide derived from the YAC end amplified a single locus from barley genomic DNA. This is a prerequisite for genetic mapping. Cultivar Ingrid
Using the genomic DNA of (1) and the source DNA of the YAC library, PCR analysis was performed using each YAC end-specific primer pair. The primer corresponding to Y113L is YAC Y
73 also revealed multi-amplicons of different lengths consistent with the non-specific PCR products observed.

In contrast, the oligonucleotides corresponding to the YAC ends Y18L, Y18R, Y30L, Y30R, Y31R, Y73R and Y113R (Table 3) amplified fragments of uniform size.
However, cloning and sequencing of the PCR products (three independent clones for each YAC end) reveal that Y30L and Y73L generate at least three different amplicons, exhibiting about 5% sequence divergence. Was done. As shown by this, the primers corresponding to Y30L and Y73L recognize multiple loci with a high degree of sequence similarity.

Sequence analysis was used to select endonucleases that recognize nucleotide stretches that are polymorphic between the three characterized subclones and sequences derived from the YAC terminus. Restriction digestion of PCR products using these diagnostic endonucleases resulted in more complex binding patterns than expected for amplicons derived from a single locus of known sequence. Thus, endonuclease-based analysis of PCR products from genomic DNA confirms the heterogeneity of amplification products corresponding to Y30L and Y73L, and in general, certain markers detect single-copy loci It is a useful tool to decide whether to do. Sequence analysis of the subclones corresponding to the YAC ends Y18L, Y18R, Y30R, Y31R and Y113R indicated that these amplicons were homogeneous.

Assignment of YAC Terminus to Barley Chromosome To determine the chromosomal location of the isolated YAC terminus, a wheat / barley diteleosomic addition lines were used, each of which was a barley cultivar Betzes. (Shepherd and Islam 1981; Islam 1983). A ditereosomic wheat / barley addendum facilitates the rapid assignment of a given barley sequence to its corresponding chromosome if the barley-specific signal can be distinguished from the wheat-specific signal.

Using PCR primers from barley cultivar Ingrid, Y18R and Y18L were assigned to barley chromosome 2HL, Y31R to chromosome 5HS, and Y73R to chromosome 6HS. This showed the chimerism of YAC Y31 and Y73. YAC-terminal Y113R could not be mapped because primers derived from cultivar Ingrid did not amplify fragments from cultivar Betzes, an additional line barley DNA donor. Similarly, the YAC-terminal Y30R could not be assigned because it generated fragments of the same size in wheat and barley.

High Resolution Genetic Mapping of YAC Ends Although the wheat / barley ditereomic addition strain facilitates the identification of chimeric YACs, high resolution genetic mapping is required to position the YAC ends relative to the target locus. is there. A prerequisite for genetic mapping of the YAC terminus is sequence polymorphism between the parental genotypes of the mapping population. A PCR-based approach was used to search for possible sequence polymorphisms. Oligonucleotides corresponding to Y30R generated amplification products of different sizes in the resistant parent (Rar1) and the susceptible parent (rar1), whereas the primer pair corresponding to Y113R amplified the DNA fragment only in each of the resistant parents.

PCR from resistant and susceptible parents for marker loci Y18L and Y18R
The products were analyzed for DNA polymorphism by direct sequencing. Comparative sequence analysis is Y
The polymorphic HinfI site was revealed in the case of 18R, but in Y18L, about 2.5 kb
No DNA polymorphism was detected over time. The copy of the 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 terminus localized Y30R and Y113R proximal to Rar1, separated by 11 and 3 recombinants, respectively, from the target locus.

YAC contig in Rar1 (i) mapping of presence / absence on YAC, (ii) chromosome assignment via additional wheat / barley lines, and (iii) information obtained by high resolution genetic mapping YAC was estimated based on YAC Y18 appears to be the only YAC containing a non-chimeric insert that is colinear with the source DNA. Because both ends map to chromosome 2HL. In contrast, YAC Y30 probably undergoes rearrangement, leading to an internal deletion containing the marker Y113R.

This conclusion is based on: (i) the genetic mapping of Y113R between MWG87 and Y30R, and (ii) the absence of a marker in YAC Y30 (Table 2). YAC by Southern analysis
Previously detailed characterization of the library revealed multiple YAC-derived fragments for about 30% instability of the clone (Simons et al., 1997). However, recovery of individual clones containing only the longest insert was usually possible. If the library still contained the corresponding clone of Y30, which also contained Y113R, the pool of yeast DNA originally used to identify the YAC clone was analyzed. YAC
No Y113R-specific amplicon could be found in the DNA of the pool corresponding to Y30, indicating that an internal deletion in YAC Y30 had occurred at an early stage during library construction.

The YAC insert chimerism was concluded for YAC Y31, Y73 and Y113. In the case of YAC Y31 and Y73, this assumption is based on chromosomes 5HS and 6HS, respectively.
To Y31R and Y73R. In contrast, evidence for chimerism in YAC Y113 is based on the absence of all markers distal to MWG87 and the fact that Y113L was detected only in YAC Y113 (Table 2). Alternatively, the YAC-terminal Y113L can be located distally from the YAC-terminal Y31L,
It means that the region between 18R and Y30L was deleted in YAC Y113 during clonal expansion.

In summary, the genomic region containing Rar1 is genetically bounded by Y113R (proximal) and MWG876 (distal). The presented YAC contig physically maps this interval by YAC clones Y18 and Y113 in the proximal orientation (2-fold redundancy) and by YAC clones Y30 and y31 in the distal orientation (2-fold redundancy). The Rar1 locus was physically demarcated in order to cover it.

Construction of BAC Contig in Rar1 Next, two HindIII BAC sublibraries were established in the vector pBelo BAC11 from YAC clone Y18 and YAC clone Y30. We aimed for an average insert size of 50 kb and nearly 5-fold redundancy for each sublibrary. CAPS MWG87, co-separating 5 BACs from YAC Y30 and Y18 with Rar1
(BAC 12, BAC 1J6, BAC 4C20, BAC 1G12, and BAC 3H
6). BAC 1H1 was isolated using PCR primers for the marker Y113R.

The insert size of the identified BAC was measured by PFGE. The terminal fragment of each BAC insert was isolated by inverse PCR and then sequenced. Based on the terminal sequence of BAC 4C20, a new codominance was induced, labeled EDDA (Table 4), and sequence polymorphisms between the parent genotypes of the mapped population were detected. When analyzing four recombinants within the interval MWG876-Y113R, EDDA was located proximal to Rar1. BAC 4C2
Since 0 contains each of the three loci MWG87, Y18R, and EDDA, Rar1 was physically bounded in the direction of the centromere on a single BAC clone.

Subsequently, the terminal sequences of BAC 12, BAC 3H6 and BAC 1B2 were used to derive markers OK1114, OK3236 and OK5558, respectively (Table 3). By examining genomic DNA from four recombinants within the target interval MWG876-Y113R, the co-dominant marker OK1114 was found to co-segregate with Rar1. By the markers OK3236 and OK5558, the parental genotype Sultans5 / Mla-12 BC Pallas
And a polymorphism between Sultans5 / Mla-12 BC Ingrid was detected,
This locus could not be detected between / Mla-12 BC Siri.

Therefore, to localize recombination events, target MWG876-Y113
Only three of the four recombinants in R could be tested. Analysis of the DNA of these three recombinants revealed cosegregation of markers OK3236 and OK5558 with Rar1. A high resolution genetic map at the Rar1 locus was estimated based on the BAC insert length, the presence and absence of each of the aforementioned markers, and their genetic orientation with respect to Rar1. Rar1 was physically demarcated by identification of BAC levels in the orientation of the centromere and the minimum cosegregation interval bounded by markers Y18R and OK5558.

It was decided to assemble adjacent genomic DNA at the DNA interval bounded by the adjacent 66 kb DNA stretch marker MWG87 and OK5558 at the Rar1 locus. BAC 1B2 and BAC 12 inserts covering this region were DNA sequenced by randomly selected clones from each BAC plasmid sublibrary into the vector pBluescript II Ks +. The 49 bp gap between BAC B2 and BAC 12 was closed by using primers specific for the terminal sequence of each BAC on template BAC 3H6.

Search for Candidate Genes Next, using the BLAST algorithm and all available databases (including ESTs of unknown author) and genomic sequences as well as known gene and protein sequences, we The search for candidate genes in the 66 kb DNA stretch was started. We have identified three distinct regions containing sequences that are highly homologous to various accession sequences in the database (Table 5). We refer to these three intervals as I1, I
2, and I3 (corresponding to positions 43,500-45,000, 45,001-46,500, and 56,000-61,000 in the 66 kb sequence contig).

Intervals I1 and I2 are related sequences (59% nucleotide identity) and were identified by the same class of ESTs in the database (Table 5), each showing similarity to the aquaporin gene. [Maurel, 1997]. That is, sections I1 and I
2 is two putative barley quaquaporin genes arranged from head to tail. Interval I3 shows high sequence similarity to rice EST C28356 and may be another coding region in a 66 kb stretch.

Identification of Mutation Events in Rar1 Candidate Genes We next examined the genotypes Rar1 Sultan5, rar1-1 Sultan5, and rar1-2 Sultan5.
Of genomic amplicons (covering sections I1, I2, and I3, respectively)
DNA sequences were compared. No sequence polymorphism was detected between the three genotypes in intervals I1 and I2. However, two single nucleotide substitutions were found at 56,764 and 58,562 base pairs in interval I3.

G → A substitutions correspond to unique sequence changes in genotypes rar1-1 and rar1-2, respectively. Since a mutation event was found in I3, we then used a set of primers deduced from the I3 sequence, using total RNA from leaves from cultivar Ingrid to perform reverse transcription polymerase chain Reaction (RT-PCR) was performed (Y
Both AC and BAC recombinant clones contain genomic DNA from the cultivar Ingrid).

Sequencing of the largest RT-PCR product revealed a single large reading frame of 696 bp (FIG. 1). Using the rapid cDNA end amplification (RACE) method, the 5 'and 3' ends of the gene transcript were identified. This putative protein of 232 amino acids has a molecular weight of about 25.5 kDa. No significant homology was found to other characterized proteins in various databases. Comparison of the genomic sequence with the sequence obtained from RT-PCR revealed six exons, each with a consensus splice site sequence flanked (Figure 2).
And Table 6). Only three pairs of consensus splice site sequences were flanked and exon 2 encoding a glycine residue was prominent.

The G → A DNA substitution identified in genotype rar1-1 Sultan5 causes a Cys ²⁴ → Tyr substitution in the putative 25.5 kDa protein (Cys ²⁴ is one of the few invariant amino acids in the Rar1 cognate protein. Below). In contrast, genotype rar1-2 Sultan
The G → A DNA substitution identified in 5 destroys the 3 ′ splice site consensus sequence of intron 2.

The G nucleotides of the splice site consensus are found in both plants and mammals.
It is known that it is essential for efficient splicing of the primary mRNA transcript [Good
all, 1991]. RT-PCR analysis of the rar1-2 genotype indicates that the mutation uses a hidden splice site in exon 3 (a phenomenon documented in many human inherited disorders caused by point mutations). [Krawczak, 1992]
[Brown, 1996]. The use of a hidden splice site shifts the reading frame and creates a new stop codon, thus causing a truncation of the putative 25.5 kDa protein.

The finding of single base substitutions in genotypes rar1-1 and rar1-2 suggests that sodium azide, a mutagen originally used for mutagenesis in cultivar Sultan5. Mode of operation. rar1-1 and rar1-2 of chemically population mutagenized used to identify, in a single gene, 0.5 × 10 comparable to the average efficiency of chemical mutagenesis in barley ^- Mutations with functional defects at ^three frequencies (Torp and Jorgensen 1986) were revealed.

[0169] Thus, two genes in a single gene that have their function in two independent mutants
The probability of identifying one point mutation is about 0.25 × 10 ⁻⁶ . In conclusion, two mutational events in genotypes rar1-1 and rar1-2 cause invariant single amino acid substitutions or splice site defects in candidate genes in physically bounded target intervals. The findings fully indicate that we isolated Rar1.

Homologs of barley Rar1 DNA sequencing of rice EST C28356 of unknown creator revealed a putative Rar1 of 233 amino acids.
A single large reading frame of 699 base pairs encoding a homologous protein has been demonstrated,
75% DNA similarity and 86% amino acid similarity to barley Rar1 were revealed (Figure 3).
) (GCG program GAP, gap creation penalty = 3 and gap extension pena
lty = 0.1). A further search of the Arabidopsis thaliana genome database revealed a section of the genome on chromosome 5 that was highly sequence related to barley and rice Rar1.

Stretches that are homologous in sequence are restricted to the exons identified in barley Rar1, and their order in the Arabidopsis genome is consistent with that identified in barley. Each of the homologous stretches of the sequence in Arabidopsis is flanked by consensus splice site sequences. This made it possible to derive a putative homologous Arabidopsis Rar1 protein of 226 amino acids with 67% identity and 75% similarity to barley Par1 (GCG program GAP, gap creation penalty = 3 and gap extension pena
lty = 0.1). We name the corresponding rice and Arabidopsis genes OsRarl1-h1 and AtRar1-h1, respectively.

Primers AtRar1 5'-ACTCCTACCTTCTCAATTCGTCCG- and AtRar1 3'-TATCAG
ACCGCCGGATCAGG- (corresponding to AtRar1-h1) allowed us to isolate a homologous cDNA, which confirmed the predicted intron-exon structure. Finally, we have studied humans, mice, Drosophila, Caenorabditis elegans, Aspergillus (A
spergillus), identified many ESTs, which are expressed genes of unknown function, and revealed significant homology only at the predicted amino acid level.

The relationships found between these ESTs and the putative protein corresponding to barley Rar1 are shown in Table 7, and the alignment of the putative sequences is shown in FIG. Obviously Barley Rar1
Indicates a novel protein domain shared among multicellular organisms. Interestingly, Cys ^{24 of} barley Rar1 (substituted for Tyr in mutant rar1-1) is one of the few amino acids that is strictly conserved in proteins related to all identified sequences. It is.

Discussion We have now described the molecular isolation of barley Rar1 by a map-based cloning approach. This gene is predicted to encode a novel 22.5 kDa intracellular protein. This finding suggests that Rar1 may have different race-specific resistance genes (
R gene) and should be interpreted in view of genetic data, which indicates that it is essential for many powdery mildew resistance reactions. It is generally recognized that plant resistance to a particular pathogen involves a specific recognition event initiated by the corresponding R gene in the host and a non-pathogenic gene (Avr) in the pathogen.

A number of effector components are involved in pathogen inhibition of pathogens, including reactive oxygen species, activation of phytoalexins, accumulation of disease-associated proteins, and cross-linking of plant cell walls. Many of these responses have also been demonstrated to be activated by barley / mildew interactions. Several types of R activated by tolerance response
Since Rar1 is required for the gene initiation resistance reaction and many effector components,
We believe that the Rar1 protein acts "downstream" of R gene recognition but acts "upstream" of executing the response. That is, Rar1 may be the convergence point of R-initiated resistance signaling.

Close examination of the Rar1 protein sequence and comparison of rice and Arabidopsis homologs reveals a prominent three-part structure (designated domains I, II, and III, FIG. 6). Interestingly, domains I and III (each about 60 amino acids in length and located near the amino and carboxy termini of Rar1, respectively) are structurally related to each other. The strictly conserved patterns of cysteine and histidine in domains I and III are prominent.

This not only preserves domain features among the plant Rar1 homologs, but also
, Aspergillus, Drosophila, Kaeno
Identification of proteins from Caenorabditis, mouse and human
It is also found in each of the other related peptide stretches. We have this
The new protein domain was named “CHORD” (in the example of domain I of Rar1, “CHORD”
ORD1 "and in the domain III example," CHORD2 "). A characteristic set of systems
Independently of in and histidine residues, CHORD is a slightly invariant amino acid Gly^{twenty three}, Phe^{Four 7} , And Trp⁵⁴, And the negatively charged residue at position 49 and the positively charged residue at position 52
(Number refers to amino-terminal CHORD domain in barley Rar1
Do).

The conservation of CHORD in such diverse species indicates a selective pressure to maintain a similar three-dimensional structure in which cysteine and histidine residues play a major role. A series of cysteine and histidine residues conserved in the intracellular protein domain have often been shown to be involved in binding zinc ions. However, the pattern of these residues in CHORD is clearly distinguished from the traditional zinc-binding domain, in which the zinc ion is a small, self-folding and functional protein domain (eg, TFIIIa zinc finger, GAL4 zinc finger). , A zinc binding domain, an LIM domain, a RING finger domain, and a GATA-1 finger domain) in the estrogen receptor.

CHORD domains (eg, CHORD1 and CHORD2) are represented as Cx ₄ -Cx _10-13 -Cx ₂ -Hx ₆ -y ₁ -x _6-7 -C ₂ -x ₁₅ -Cx _4-5 -H And Cx ₄ -Cx ₁₃ -Cx ₂ -Hx ₆ -Hx ₇ -C ₂ -x ₁₅ -Cx ₄ -H.

In particular, the CHORD domain of the invention has the formula: Cx ₃ -GCx ₃ -A ₁ -x _6-9 -Cx ₂ -Hx ₅ -Fy ₁ -A ₂ -x _1-2 -A ₃ -x ₁ -Wx ₁ -CCx ₁₅ -Cx _4-5 -H (where C, G, F and W are _one- letter codes of Cys, Gly, Phe and Trp, respectively, A ₁ is an aromatic amino acid, Phe, is selected from Trp and Tyr, a ₂ is a residue which is negatively charged, is selected from Glu and Asp, a ₃ is charged residues positive, Arg, is selected from His and Lys , Y ₁ is H or any amino acid, preferably His or Arg, x is any amino acid (the number indicates the number of amino acids), and cysteine and histidine required for zinc binding Subject to the structural limitations of the spatial relationship.

The domain III in the Rar1-like protein is a domain of the present invention (Cx ₂ -Cx ₅ -Cx ₂ -H
) Appears to contain another set of cysteine and histidine residues. It binds to a protein encoded by the Arabidopsis homolog of the yeast SGT1 gene (Kitagawa et al. (1999) Mol Cell 1: 21-34).

The molecular isolation of barley Rar1 and the finding of related genes in rice and the dicotyledon Arabidopsis thaliana may be a component of all higher plant genomes. The degree of sequence conservation in the Rar1 homolog of plants indicates that they may share related functions. Only a 2-3 fold overexpression of the Arabidopsis thaliana NPR1 gene, a major regulator of systemic acquired resistance, is associated with the pathogen Peronospora paracitica (
Peronospora parasitica) and Pseudomonas syringae
(Cao, 1998), indicating that modulating the steady-state level of Rar1 mRNA or protein can be used to alter the rate of response and the pathogen spectrum.

This gene was fused to a promoter from a pathogenicity-related gene (PR gene) to
Redirecting r1 expression can also be used to broaden the spectrum of Rar1-mediated pathogen resistance. This approach is particularly attractive when combined with the expression of derivatives of the Rar1 protein. For example, a variant of the Rar1 protein that uncouples activation from the R gene and retains activation of downstream responses (PR gene activation, HR) is identified. The identified three-part domain structure of the plant Rar1 protein may be useful in guiding these experiments.

Additional Materials and Methods for High-Resolution Genetic and Physical Mapping Plant material doubled haploid barley (Hordeum vulgare) strain Sultan-
5 and Sultan-5 derived mutants M82 (rar1-1) and M100 (rar1-2)
And Jorgensen 1986. Sultan-5 and mutants contain the macroscopic marker gene ant2 (deficient anthocyanin in leaf sheath). The Mla-12 backcross (BC) strain in the cultivars Siri and Pallas has been described by Kolster et al., 198
6; described in Kolster and Stolen et al., 1987.

The Mla-12 BC strain in the cultivar Ingrid was H. vu
lgare) was produced by seven backcrosses on the cultivar Ingrid followed by at least seven selfings. Each mutant M82 and M100, pollinate the pollen from MLA-12 BC strain cultivated, grown to maturity F ₁ plants from the cross, to obtain a variety of separate F ₂ population.

Crossbreeding Nipponbare x Kasalath (Kurata et al., 1994)
186 individuals separate F ₂ population obtained from a Mapping of rice. The map location of the rice locus MWG876 was tested independently in a _second isolated F2 population of 123 individuals obtained from the cross IR20 × 63-83 (Quarrie et al., 1997).

Testing for Resistance Testing for resistance was performed as described in Freialdenhoven et al., 1994. In F ₃ and F ₄ in families selfing and after subsequent inoculation experiments comprising at least 25 individuals, was determined the phenotype of recombinant. The F ₃ individuals were tested by polymorphic sequence analysis cleavable (CAPS), it was identified homozygous recombinant. These plants selfed again, were resistance test at F ₄ family. Seven days after inoculation, plants were scored for resistance / sensitivity.

Pulsed Field Gel Electrophoresis (Pfge) and Southern Analysis Barley high molecular weight DNA was obtained using the method of Siedler and Graner (1990) for 5-7 minutes.
Isolated from aged seedling leaf material. Using the protocols of Ganal and Tanksley (1989), six rare cleavage restriction enzymes (ClaI, MluI, SalI, NotI, SfiI, SfiI, SgfI, Sma
The DNA was digested using I). For size fractionation, LKB Pulsaphor®
Equipment (Pharmacia Biotech), Uppsala,
Swedish) 180V at pulse time of 10-60s (linear interpolation method), 25 hours, 0.5 × T
A 1.2% agarose gel was run at 12 ° C. in BE (50 mM Tris-HCl, 50 mM boric acid, 1 mM EDTA, pH 8.3). Capillary migration and non-radioactive Southern hybridization were performed as described by Lahaye et al. (1996).

AFLP / CAPS Analysis Genomic DNA was isolated for CAPS and AFLP analysis according to Stewart and Via (1993). Table 1 shows the primer PCR conditions for CAPS marker display and each restriction enzyme. CAPS analysis was performed using a 20 μl volume (100 pmole of each primer, 200 μM dNTP, 10 mM Tris-HCl, pH 8.3, ₂ mM MgCl ₂ , 50 mM KCl ₂ , 0.5 U Taq polymerase (Boehringer)) and 50 ng of barley genome template. Performed using DNA.

The digested PCR product was then serially size fractionated on a 2% agarose gel. AFLP
The analysis (Vos et al., 1995) analyzed the resistant and susceptible plants (Giovannoni et al., 1991; Michelm
ore et al., 1991) using PstI and MseI restriction enzymes, PstI and MseI adapters, and two or three alternative nucleotides at the 3 'end, respectively.
Performed using one set of primers corresponding to the MseI adapter. 7 Psts
A total of 392 combinations were analyzed using I + 2 (2 alternative bases)-and 56 MseI + 3 (3 alternative bases) -primers.

Additional Materials and Methods for Analysis of YACS Each of the 96 yeast clones (Simons et al., 1997) using barley YAC library screening CAPS marker MWG87.
The DNA of 28 YAC pools was screened. The YAC pool positive for the MWG87 amplicon was further analyzed by colony PCR for identification of a single yeast clone. Observed amplicons were size-separated on agarose gel, cut, and
k-gel extraction kit (Quiagen GmbH, Dusseldorf, Germany)
Was purified.

The purified PCR product was used for dye terminator sequencing (Perkin Elmer
kin-Elmer) and Norwalk, Connecticut, USA) and confirmed that the amplicons generated corresponded to a single copy locus of MWG87. Next, each of the observed YAC clones was examined with the CAPS marker MWG876. The cycling conditions and primers used for the CAPS markers MWG87 and MWG876 were
It is described herein. The initial denaturation step was extended from 2 minutes to 4 minutes for colony PCR analysis.

The seeds of the plant material diteleosomic wheat / barley supplemented strain were Shephe
rd and Islam 1981; as described in Islam 1983.

CAPS Analysis Plant DNA for PCR-based analysis was extracted according to Stewart and Via 1993. Y
Table 4 shows the primers and the PCR conditions of the AC terminal specific marker. PCR was performed in a volume of 20 μl (100 pmole of each primer, 200 μM dNTP, 10 mM Tris-HCl, pH 8.3, 2 mM MgC
l ₂ , 50 mM KCl ₂ , 0.5 U Taq polymerase (Boehringer Mannheim (Boehringer
Mannheim, Mannheim, Germany) using 200/50 ng of wheat / barley genomic template DNA. Amplicons corresponding to different YAC ends were cloned into the pGEM-T vector (Promega, Southampton, UK) and three independent clones of each PCR product were dye terminator sequenced (Perkin Elmer). -Elmer)).

PFGE Southern Analysis of YACS Individual YAC clones were grown at 30 ° C. for 2 days at 30 ° C. in 100 ml of synthetic dextrose (SD) minimal medium lacking uracil and tryptophan (Rose et al., 1990). High molecular weight yeast DNA was prepared in low melting point agarose as described by Carle et al. (1985).

Separation of yeast chromosomes was performed at 180 V in an LKB Pulsaphor® instrument (Pharmacia Biotech, Uppsala, Sweden) at 10OV.
30 hours, 0.5 × TBE (50 mM Tris-HCl,
This was performed on a 1.2% agarose gel at 12 ° C. in 50 mM boric acid, 1 mM EDTA, pH 8.3).
Capillary migration and non-radioactive Southern hybridization were performed as described by Lahaye et al. (1996). The size of the YAC insert was then determined using MWG87 as a probe for Southern hybridization as described by Lahaye et al. (1996).

Isolation of YAC Terminal Sequence The terminal sequence of the YAC insert was isolated by reverse PCR (IPCR) of Silverman et al. (1989). The observed amplicons were size-separated on agarose gel, cut, and
Purification was performed with an ick gel extraction kit (Quiagen), and dye terminator sequencing (Perkin-Elmer) was performed. Additional IPCR at YAC end
A new oligonucleotide was deduced based on the sequencing information at each YAC end for further extension.

Database Search Analysis of YAC terminal sequences was performed using the Unix users Genetics Computer Groups (GSG) and the STADEN software package program (4th edition, 1994).

Embodiment 2 FIG. Downstream activation of death flashing of-independent Rar1 dependent host cells R gene induction in barley Ubi1- intron 1 (Wan and Lemaux, 1994) known maize ubiquitin promoter is located laterally (Ubi1 promoter), Overexpression of the full-length and truncated Rar1 gene derivatives of barley was performed using standard gene cloning methods (Sa
mbrook et al., 1989).

[0200] Briefly, the vector pUBI-GFP (Carlsberg Research Laboratory, Copenhagen, Denmark) contains the RAR-1 gene of interest (eg, the entire RAR-1 gene, or It is modified by inserting a sequence encoding its N-terminal or C-terminal portion, such as those shown in FIGS. 5A and 5C, respectively, encoded by the sequences of FIGS. 5B and 5D. After cloning of the constructs, they are administered and leaves of 7-day-old plants are prepared from barley using a suitable transient transformation protocol.

Plant Material for Transient Transformation Seeds of Golden Promise, a Hordeum vulgare cultivar, were incubated for 1 minute in 70% ethanol,
Wash three times with li-Q water, then incubate with 1.5% sodium chloride for 10 minutes,
Wash 5 times with Milli-Q water. Sow (15 seeds / sample) in magenta containing 2 cm of vermiculite supplied with 30 ml of 1/2 strength MS basal medium (Sigma) supplemented with 2% sucrose and cultured at 22 ° C (16 hours light place / 8 hours dark place). The first leaf of a 7-day-old seedling is cut over the cotyledon sheath and cut into two 3 cm sections. The sample is incubated with 3 ml of 10% sucrose in a Petri dish for 3 hours, which causes the plant material to float. The sucrose solution is then removed and the plant material is air dried for 5 minutes before particle bombardment.

Transient Transformation Method The transformation method uses a particle flow gun (PIG) (Vain et al., 1992). The produced body is 1
1 μg of Quiagen-purified plasmid is introduced per impact and deposited on gold particles (1 μm, Bio Rad) according to the method of Klein et al. (1988). The plant material is placed 9 cm below the particle outlet and covered with steel grit with a pore size of 0.4 mm.
Impact conditions: accelerate particles with 2735 mbar helium gas at an air pressure of 100 mbar. D
Immediately after the NA supply, 4 ml of sterile 1/2 strength MS basal medium (supplemented with 3% sucrose and 1 mM benzimidazole) are added to the sample, which is then incubated at 24 ° C. in the dark for 24 hours.

Results The appearance of the dead cell population is confirmed by trypan blue staining. Leave leaf samples
Boil for 8 minutes in alcoholic lactophenol (96% ethanol-lactophenol 1: 1 [v / v]) containing 1 mg / ml trypan blue (Sigma) for 8 minutes and stain with chloral hydrate solution (2.5%). (mg / ml) overnight. Rar1 constructs that activate host cell killing by transient assays are selected for further modification in transgenic plants.

Embodiment 3 FIG. By fusion to a promoter from a pathogenicity-related gene (Pr gene)
The expressed PR gene of B cell killing, which activates Rar-1 derivatives in barley, is known to be activated at high levels around the site where pathogen attack was attempted. The cell killing activated Rar-1 derivative is fused to the 2 kb promoter sequence of the barley genes HvPR1-a and HvPR1-b (Bryngelsson et al., 1994). These genes are found in powdery mildew fungi, Drechslera teres and Pukinia hordei (
It is known to be activated in leaf tissue in response to attacks from different pathogens, including Puccinia hordei) (Reiss and Bryngelsson, 1996).

The fusion of the HvPR1-a and HvPR1-b promoter sequences to the cell killing activated Rar-1 derivative described herein replaces both the uidA receptor gene and the corn ubiquitin promoter, and then By cloning (Sambrook et al., 1989), it is cloned into the vector pAHC25 (Wan and Lemaux, 1994). Transgenic barley plants of the cultivar Golden Promise are pAHC
Using the selectable marker gene bar in 25, and then using the method of Wan and Lemaux (1994).

After inoculation with different isolates of powdery mildew, Puccinia hordei, and Drechslera teres spores, transgenic strains are tested for broad spectrum resistance. Plants exhibiting resistance to the described isolated cytokines are observed.

Activating Rar1-dependent host cell killing independent of R gene induction Ubi1-intron 1 (Wan and Lemaux, 1994) is located downstream of the known maize ubiquitin promoter (Ubi1 promoter). Overexpression of the full-length and truncated Rar1 gene derivatives of barley was performed using standard gene cloning methods (Sa
mbrook et al., 1989).

In brief, the vector pU-Mlo (pUBI-GFP was replaced with a 1.8 kb Mlo cDNA (Boschges
Shirasu K et al., 1999, Plant J., 17 (3): 293-299), except for Mlo, where the RAR-1 gene of interest (eg, the entire RAR-1 gene or its N-terminus) 5A and 5C, respectively, which is encoded by the sequence encoding the portion or the C-terminal portion, such as those shown in FIGS. After cloning of the constructs, they are administered and leaves of 7-day-old plants are prepared from barley using a suitable transient transformation protocol (see Example 2).

Results The appearance of the dead cell population is confirmed by trypan blue staining. Leave leaf samples
Boil for 8 minutes in alcoholic lactophenol (96% ethanol-lactophenol 1: 1 [v / v]) containing 1 mg / ml trypan blue (Sigma) for 8 minutes and stain with chloral hydrate solution (2.5%). (mg / ml) overnight. Rar1 constructs that activate host cell killing by transient assays are selected for further modification in transgenic plants.

Embodiment 4 FIG. Expression of the cell killing activated Rar-1 derivative in dicotyledonous plants independently of the R gene (as in Example 2 for barley) and a pathogenicity-related gene (Pr gene) (as in Example 3 for barley) (2) by fusion to a promoter. The preparation of the plant material is carried out as described above for barley using surface-sterile Arabidopsis (ecotype Columbia) and tomato seeds (Moneymaker). Agrobacterium containing the p35S-Rar1 construct (Agroba
cterium) strain C58 (Rar1 driven by the p35S CaMV promoter in the T-DNA vector pBIN19 (Bevan, 1984)).

Results The appearance of the dead cell population is confirmed by trypan blue staining. Leave leaf samples
Boil for 8 minutes in alcoholic lactophenol (96% ethanol-lactophenol 1: 1 [v / v]) containing 1 mg / ml trypan blue (Sigma) for 8 minutes and stain with chloral hydrate solution (2.5%). (mg / ml) overnight. Rar1 constructs that activate host cell killing by transient assays are selected for further modification in transgenic plants.

Embodiment 5 FIG. Cell Killing Activated At-Rar1 Derivative in Arabidopsis
The glucocorticoid-mediated transcription induction system is used for inducible overexpression of full-length and truncated AtRar1 gene derivatives in Arabidopsis. Using the XhoI and SpeI cloning sites, insert the desired AtRar1 sequence (the entire AtRar1 gene, which is a sequence encoding the N-terminal, internal, or C-terminal portion) selected from those shown in FIGS. 8A-8H Thereby modifying the vector pTA231 (Rockefeller University, New York, USA). Primers used to amplify the AtRar1 gene fragment are:

OK228 5'-CCTCGAGACTCCTACCTTCTCAATTCGTCCG-3 'and OK232 5'-AACTAGTATCAGACCGCCGGATCAGG-3' for all AtRar1, OK228 and OK229 5'-AACTAGTCAGGCCAGAACTGGTTTCTCAGTTGT-3 'for N-terminal part
For the internal part, OK230 5'-AACTAGTCAAGCCTTTTGTACTGGAGGCGC-3 'and OK231 5'-ACTCGAGATGGCCAAATCGGTTCCAAAACATC-3' and for the C-terminal part, OK233 5'-ACTCGAGATGGCTGTGATAGACATTAATCAACCGC-3 'and OK23
2.

A standard Arabidopsis transformation protocol (Clough and Bent)
Arabidopsis, 1998, Plant Journal 16, 735-743).
Plants are transformed with Agrobacterium strain C58 containing the above construct.

References Anderson et al. (1997). The Plant Cell 9, 641-65l. Bennett, MD and Smith, JB (1991). Nuclear DNA content in angiosperms, Philosophical
Transactions of the Royal Society Of London B Biological Sciences (Londo
n: Royal Society), pp. 309-345.Bent et al. (1994) .Science 265, 18S6-1860.Bevan M. Nucleic Acids Research, 1984, Vol. 12, N0.22, pp. 8711-872l Bickmore, WA , And Bird, AP (1992) .Methods In Enzymology 216, 224-24
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2, 6597-6601

Century et al. (1997). Science 278, 1963-1965 Devereux et al. (1984). Nucleic Acids Res. 12, 387-395. Dixon. RA, and Lamb, CJ (1990). Annual Review Of Plant Physiology and
Plant Molecular Biology 41, 339-367. Flor, HH (1971) .Anual Review of Phytopathol0gy 9, 275-296. Freialdenhoven et al. (1994) .The Plant Cell 6, 983-994. Giovannonl et al. (1991) .Nucleic Acids Res. 19, 6553-6558. Goodall, GJ, and Filipowicz, W. (1991). EMBO Journal 10, 2635-2644. Graner et al. (1991). Theor Appl Genet 83. 250-256. Grant et al. (1995). Sciencie 269. , 843-846

Hammond-Kosack et al. (1994). The Plant Cell 6, 361-374. Hammond-Kosack, KE, and Jones, JDG (1996). Plant Cell 8, 1773-1791. Islam, AKMR (1983). Ditelosomic. Additions Of Barley Chromosomes T
o Wheat. In Proc. 6th International Wheat Genetics, Kyoto, Japan, pp. 23
3-238. Jones et al. (1994). Science 266, 789-793. Jorgensen JH (1996). Genome 39, 492-498. Jorgensen. JH (1988). Genome 30, 129-132. Klein TM et al. (1988). Proc. Natl. Acad. Sci. USA 85, 4305-4309.

Kolster et al. (1986) Crop Science 26, 903-907 Kolster P., and Stolen. O. (1987). Plant Breeding 98, 79-82. Krawczak et al. (1992). Human Genetic 90, 41-54. Kurata et al. (1994) .Nature Genetics 8, 365-372. Lahaye et al. (1996) .BioTechniques 21, 1067-l072. Larsen et al. (1992) .Genetic Analysis Techniques and Applications 9, 80-85.
Lawrence et al. (1995) .The Plant Cell 7 1l95-1206. Martin et al. (1993) .Science 262, 1432-1436.Maurel, C. (1997) .Annual Reviews of Plant Physiolog and Plant Molecular.
Biology 48, 399-429.

Michelmore et al. (1991). Proceedings of the National Academy of Sclences, US
A 88, 9828-9832. Mindrinos et al. (1994) .Cell 78, 1089-l099. Ori et al. (1997). The Plant Cell 9, 521-532. Parker et al. (1996). The Plant Cell 8, 2033-2046. Peterhansel et al. (1997) .Plant Cell 9, 1397-1409. Quarrle et al. (1997) .Plants Mol Biol 35, I55-165. Relss E. & Bryngelsson T. Physiological and Molecular Plant Pathology, 1
996.Vol.49, No.5, pp.331-341.

Rose et al. (1990) Methods in Yeast Genetics. Cold Spring Harbor Laboratory,
Cold Spring Harbor, New York, USA. Russel et al. (1997). Theoretical and Applied Genetics 95, 714 -722. Salmeron et al. (1994). The Plant Cell 6, 5ll-520. SanMiguel et al. (1996). Science 274, 765. -768. Shepherd, KW, and Islam. AKMR (1981). Wheat: Barley Hybrids-The
First Eighty Years. In Wheat science-today and tomorrow, LT Evans
WJ Peacock, eds. (Cambridge: Cambridge University Press), pp. 107-12
8.

Natl. Acad. Sci. USA 86, 7485-7489. Simons et al. (1997). Genomics 44, 61-70. Song et al. (1995). Silence 270, 1804-1806. Suoniemi et al. (1996) .Plant Molecuar Biology 30, 1321-1329.Torp, J., and Jorgensen, JH (1986) .Canadian Journal of Genetics and Cyt.
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95, 1663 1668

[0223]

[Table 1]

[0224]

[Table 2]

[0225]

[Table 3]

[0226]

[Table 4]

[0227]

[Table 5]

[0228]

[Table 6]

[0229]

[Table 7]

[Brief description of the drawings]

FIG. 1 shows the nucleotide and deduced amino acid sequence of barley Rar1 cDNA. The nucleotide and deduced amino acid sequences are based on the combined RT-PCR and RACE data obtained from experiments using the cultivar Ingrid Rar1. The stop codon is marked with an asterisk, and the detected end of the RACE product is indicated by an arrow above the sequence.

FIG. 2 illustrates the structure of the Rar1 gene. The structure of the barley Rar1 gene is given schematically. Exons are highlighted by black boxes. Intron and exon positions were identified by comparison of the RT-PCR product with the genomic sequence. For mutations rar1-1 and rar1-2, the location of the mutation event is indicated.

FIG. 3 shows the alignment of deduced peptide sequences in genes from various species,
The relationship to barley Rar1 is shown. Regions of homology are highlighted in black (identity), dark grey (highly conservative exchanges) or pale grey (conservative exchanges).
Sequence data is transferred to Genetics Computer Group (Genetics Comp
(uter Group, Wisconsin Program) version 8 (GCG; Devereux, 1984). In the extended GCG software, "prettybox"
A) Display of the aligned and deduced amino acid sequence was performed by using the ")" option. The numbers on the left indicate the accession numbers of the gene banks for each peptide sequence.

FIG. 4 shows the 10,000 nucleotides of the Rar1 genomic gene sequence, including the coding exons and introns. rar1-1 and rar1-2 mutations (both G-> A)
Mark. The underlined sequence represents the Rar1 exon sequence, and the boldface nucleotides represent the 5 'and 3' consensus splice sequences.

FIG. 5A shows the amino acid sequence of the N-terminal fragment of the Rar1 polypeptide of FIG.

FIG. 5B shows the nucleotide sequence encoding a fragment of the Rar1 polypeptide of FIG. 5A.

FIG. 5C shows the amino acid sequence of the C-terminal fragment of the Rar1 polypeptide of FIG.

FIG. 5D shows the nucleotide sequence encoding a fragment of the Rar1 polypeptide of FIG. 5C.

FIG. 6 shows fragments of barley Rar1 protein and domains I, II and
3 shows the amino acid sequence of III and represents a particular aspect of the invention.

FIG. 7 shows the fragments of FIG. 6 and the nucleotide sequences encoding domains I, II and III, polynucleotides having these sequences, and polynucleotides comprising these sequences. Represents the other side of

FIG. 8A shows the AtRar1 cDNA sequence, including the coding sequence.

FIG. 8B shows the AtRar1 protein sequence (also shown as ab010074 in FIG. 3).

FIG. 8C shows the coding nucleotide sequence for the N-terminal fragment of the AtRar1 protein.

FIG. 8D shows the AtRar1 protein encoded by the nucleotide sequence of FIG. 8C.
The N-terminal fragment is shown.

FIG. 8E shows the coding nucleotide sequence for an internal fragment of the AtRar1 protein.

FIG. 8F shows an internal fragment of the AtRar1 protein encoded by the nucleotide sequence of FIG. 8E.

FIG. 8G shows the coding nucleotide sequence for the C-terminal fragment of the AtRar1 protein.

FIG. 8H shows the AtRar1 protein encoded by the nucleotide sequence of FIG. 8G.
The C-terminal fragment is shown.

FIG. 9 shows the alignment of various “CHORD” (“cysteine and histidine rich domains”) and consensus sequences.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12Q 1/68 C12R 1:91) G01N 33/53 C12N 15/00 ZNAA // (C12N 5/10 5 / 00 C C12R 1:91) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP , KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Shirasu, Ken United Kingdom, Norwich N.R. 23 JH, Glebe Road 107 (72) Inventor Raie, Thomas Germany, Day-06120 Halle, Weinburgbeek 22, Halle, Martin Luther Universitet, Institute of Genetics Fta (Reference) 2B030 AA02 AB03 CA17 CA19 CD03 CD07 CD09 CD13 CD14 4B024 AA08 BA80 CA03 CA04 DA01 EA04 GA11 HA01 HA20 4B063 QA01 QA07 QA19 QQ04 QQ43 QR32 QR55 QR62 QS05 QS34 4B065 AA88X AA01A15A15 BA15 A15A15A15A15A15BA15 EA60 FA74

Claims (38)

[Claims] 1. An isolated polynucleotide encoding a polypeptide that functions in a plant pathogen defense response signaling pathway, wherein the polypeptide comprises
The above polynucleotide comprising an amino acid sequence having at least 70% amino acid sequence identity with the amino acid sequence shown in the above. 2. The isolated polynucleotide of claim 1, wherein said polypeptide has the amino acid sequence shown in FIG. 3. The polynucleotide has the coding sequence shown in FIG.
3. The isolated polynucleotide according to claim 2. 4. An isolated polynucleotide that encodes a polypeptide that functions in a plant pathogen defense response signaling pathway, wherein the polypeptide, under stringent conditions, encodes the Rar1 encoding nucleotide sequence shown in FIG. The above polynucleotide, which selectively hybridizes with a probe which is a complement of the above. 5. An isolated polynucleotide encoding the barley Rar1 fragment shown in FIG. 5A, FIG. 5C or FIG. 6. The isolated polynucleotide of claim 5, wherein the polynucleotide has one of the coding sequence shown in FIG. 5B or FIG. 5D or the coding sequence shown in FIG. 7. The Arabidopsis shown in FIG. 8D, 8F or 5H.
2.) An isolated polynucleotide encoding a Rar1 fragment. 8. The isolated polynucleotide of claim 7, wherein said polynucleotide has a coding sequence shown in FIG. 8C, 8E or 8G. 9. The polynucleotide according to any one of claims 1 to 8,
Or (i) a polynucleotide encoding the OsRar1-h1 amino acid sequence shown in FIG. 3; (ii) a polynucleotide encoding the AtRar1-h1 amino acid sequence shown in FIG. 3; (iii) it functions in the plant pathogen defense response signaling pathway A polynucleotide encoding a polypeptide, wherein the polypeptide is OsRar1-h1 or AtR.
the above polynucleotide, which comprises an amino acid sequence having at least 70% amino acid sequence identity to the ar1-h1 amino acid sequence; (iv) a polynucleotide encoding a polypeptide that functions in a plant pathogen defense response signaling pathway; A probe wherein the polypeptide is the complement of the gene nucleotide accession number C28356 encoding Gene OsRar1-h1 or the gene bank Accession number AB010074 encoding AtRar1-h1 under stringent conditions. An isolated polynucleotide, wherein the polynucleotide selected from the group consisting of the above-mentioned polynucleotides, which selectively hybridizes; is operably linked to a control sequence for expression. 10. The coding sequence or claim, wherein the nucleotide sequence is suitable for use in antisense or sense regulation of expression of the coding sequence ("co-suppression") and is under the control of regulatory sequences for transcription. Item 20. An isolated polynucleotide complementary to a sequence of at least 50 contiguous nucleotides of a sequence complementary to the coding sequence of the nucleic acid of any one of clauses 1-19. 11. A peptide comprising a CHORD amino acid sequence corresponding to one of the following formulas:
An isolated polynucleotide encoding a tide: (i) C-x_Three-G-C-x_Three-A₁-x_6-9-C-x_Two-H-x_Five-F-y₁-A_Two-x_1-2-A_Three-x₁-W-x₁-C-C-x_Fifteen-C-x_{Four -Five} -H (ii) C-x_Two-C-x₆-C-x_Two-H (where C, G, F and W are one-letter codes of Cys, Gly, Phe and Trp, respectively.
Yes, A₁Is an aromatic amino acid and A_TwoIs a negatively charged residue and A_ThreeIs a positively charged residue and y₁Is H or any amino acid, preferably His or Arg, x is any amino acid, and the numerical value indicates the number of amino acids), and peptide (i) binds to zinc. 12. A nucleic acid vector suitable for transformation of a plant cell and comprising the polynucleotide according to any one of claims 1 to 11. 13. A host cell containing a heterologous polynucleotide or a nucleic acid vector according to any one of claims 1 to 12. 14. The host cell according to claim 13, which is a microorganism. 15. The host cell according to claim 13, which is a plant cell. 16. The chromosome has a heterologous polynucleotide in the chromosome.
A plant cell according to item 1. 17. The plant cell according to claim 16, which has two or more polynucleotides per haploid genome. 18. The plant cell according to any one of claims 15 to 17, which is contained in a plant, a plant part or a plant embryo, or a plant extract or derivative. 19. The method for producing a cell according to any one of claims 13 to 17, comprising incorporating a polynucleotide or a nucleic acid vector into the cell by transformation. 20. The method of claim 19, comprising recombining the polynucleotide and a cell genomic nucleic acid such that it is stably incorporated therein. 21. The method according to claim 19 or 20, comprising regenerating a plant from one or more transformed cells. A plant comprising the plant cell according to any one of claims 15 to 17. A part or embryo of a plant comprising the plant cell according to any one of claims 15 to 17. 24. A method for producing a plant, comprising introducing the polynucleotide or the nucleic acid vector according to claim 1 into a plant cell and regenerating the plant from the plant cell. 25. The method of claim 24, comprising sexually or asexually reproducing or propagating progeny of a plant regenerated from the plant cells. 26. A method for affecting a characteristic of a plant, comprising causing or permitting transcription from a heterologous polynucleotide according to any one of claims 1 to 11 in a cell of the plant. . 27. Use of a polynucleotide according to any one of claims 1 to 11 in the production of a transgenic plant. 28. A method for identifying or obtaining a polynucleotide encoding a polypeptide that functions in a plant pathogen defense response signaling pathway, wherein the polynucleotide according to claim 4 under stringent conditions. Or using a nucleic acid molecule that selectively hybridizes to its complement to screen for candidate nucleic acids. 29. A nucleic acid probe or primer having a nucleotide sequence of at least about 20-30 nucleotides, which is the sequence shown in the coding region of FIG. 1 or a sequence complementary thereto. 30. The pair of primers according to claim 29, which is suitable for amplifying a fragment of the coding region of the polynucleotide according to any one of claims 1 to 9. 31. A method for identifying or obtaining a polynucleotide encoding a polypeptide that functions in a plant pathogen defense response signaling pathway from a plant or a plant cell, according to claim 29 or claim 30. The above method characterized by using the nucleic acid probe or primer of the above. 32. The method of claim 31, comprising: (a) providing a nucleic acid preparation from a plant cell; (b) providing the probe of claim 29; and (c) providing the nucleic acid preparation. Contacting a probe with a probe under conditions for selective hybridization of the probe with a nucleic acid encoding the polypeptide; and (d) contacting the nucleic acid encoding the polypeptide (if present) with: Identifying by hybridization with a probe, and optionally (e) confirming the identity of the polypeptide encoded by the nucleic acid by expression in an expression system and measuring its ability to function in a plant pathogen defense response signaling pathway, The above method comprising: 33. The method of claim 31, comprising: (a) providing a preparation of nucleic acid from a plant cell; (b) providing a pair of primers according to claim 30; Contacting the nucleic acid in the preparation with the primer under conditions for performing PCR, (d) performing PCR, determining the presence or absence of the amplified PCR product, and optionally (e) in the expression system. Expression, confirming the identity of the amplified PCR product and measuring the ability of the produced polypeptide to function in a plant pathogen defense response signaling pathway. 34. An isolated polypeptide encoded by the polynucleotide according to any one of claims 1 to 9. 35. An isolated antibody comprising an antigen-binding site having specific binding affinity for the polypeptide of claim 34. 36. A polypeptide comprising the antigen-binding site of the antibody according to claim 35. 37. A method for identifying or obtaining a polypeptide according to claim 34, comprising screening a candidate polypeptide with the antibody or polypeptide according to claim 35 or 36. 38. An isolated peptide encoded by the polynucleotide of claim 11.