JP2002537840A - Novel plant genes and their uses - Google Patents

Abstract

  (57) [Summary] Homologs of the Arabidopsis NIM 1 gene involved in the signal transduction cascade leading to systematic acquired resistance (SAR) include Nicotiana tabacum (tobacco), Lycopersicon esculentum (tomato), Brassica napus (fat seed), Arabidopsis thaliana, It is isolated from Beta vulgaris (sugar beet), Helianthus annuus (sunflower) or Solanum tuberosum (pot). The present invention further relates to transformation vectors and methods for expressing NIM1 homologs in transformed plants that increase SAR gene expression and enhance broad spectrum disease resistance.

Description

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL FIELD The present invention relates to a wide range of disease resistance in plants, including the phenomenon of systematically acquired resistance (SAR). More particularly, the present invention relates to the identification, isolation and characterization of Arabidopsis NIM1 gene homologs, including a signal transduction cascade leading to systemically acquired resistance in plants.

[0002] Plants are constantly challenged by a wide variety of pathogenic organisms or organisms, including viruses, bacteria, fungi and nematodes. Because cereal plants are grown as genetically identical single farms, they are particularly vulnerable and can be severely damaged by disease. However, most plants have intrinsic defense mechanisms against their own pathogenic organisms. Natural variants for resistance to phytopathogenicity have been identified by plant breeders and pathologists and are grown in many cereal plants. These natural disease resistance genes frequently exhibit high levels of resistance or immunity to pathogens.

[0003] Systematic acquired resistance (SAR) is one component (sequence) of a complex system that plants use to protect themselves from pathogens (Hunt and Ryals, 1996;
Ryals et al., 1996). See U.S. Patent No. 5,614,395. In particular, SAR is an important aspect of the plant pathogen response because of its pathogen-induced systemic resistance to a wide range of infectious agents, including viruses, bacteria and fungi. If the SAR signal transduction pathway is blocked, the plant is susceptible to pathogens that normally cause disease, and also to certain pathogens that would not normally cause disease. (Gaffney et al., 1993; Del.
aney et al., 1994; Delaney et al., 1995; Delaney, 1997; Bi et al., 1995; M
auch-Mani and Slusarenko, 1996). These findings indicate that the SAR signal transduction pathway is essential for maintaining plant health.

[0004] Conceptually, the SAR response can be divided into two stages. At an early stage, a pathogen infection is recognized and signals are delivered (migrated) through the phloem (skin) to distant cells. The systemic signal is recognized by target cells that respond to the expression of both the SAR gene and disease resistance. The maintenance phase of SAR extends from several weeks to the overall life of the plant, during which the plant is apparently in a steady state and disease resistance is maintained (Ryals et al., 1996).

[0005] The accumulation of salicylic acid (SA) appears to be necessary for SAR signal transduction. Plants that are unable to accumulate SA by treatment of the specific inhibitor, the epigenetic repression of the amylase of phenylalanine or the transformation expression of salicylate hydroxylase, which specifically degrades SA, are associated with SAR gene expression or disease. Tolerance cannot also be induced (Gaffney et al., 1993; Delaney et al., 1994; Mauch-
Mani and Slusarenko, 1996; Maher et al., 1994; Pallas et al., 1996). SA
Has been suggested to function as an organizational signal, but recently this has been debated, and all that is known to be certain is that if SA does not accumulate, the SAR signal Means that the production is cut off (Pal
las et al., 1996; Shulaev et al., 1995; Vernooij et al., 1994).

In recent years, Arabidopsis has emerged as a model system for SAR research (
Uknes et al., 1992; Uknes et al., 1993; Cameron et al., 1994; Mauch-Mani
And Slusarenko, 1994; Dempsey and Klessig, 1995). SARs are pathogens and chemicals such as SA, 2,6-dichloroisonicotinic acid (INA) and benzo (1,2,3)
It has been shown to be activated by both thiadiazole-7-carbothioic acid S-methyl ester (BTH) (Uknes et al., 1992; Vernooij et al., 1995; Lawton
et al., 1996). After treatment with INA or BTH or pathogen infection, at least three pathogenicity-related (PR) protein genes, namely PR-1, PR-2 and PR-5, are coordinated with each other simultaneously with the development of resistance and induced. (Uknes et al., 1992, 1993).
In tobacco, the best characterized species, treatment with a pathogen or immunizing compound induces the expression of at least nine sets of genes (Ward et al.
, 1991). Transformed disease resistant plants have been created by transforming plants with various SAR genes (US Pat. No. 5,614,395).

[0007] A number of Arabidopsis mutants exhibiting modified SAR signal transduction have been isolated (Delaney, 1997). The first of these mutant strains of, so-called, Isd (l esio
ns s imulating d isease) a mutant strain and acd2 (a ccelerated c ell d eath ) (Di
etrich et al., 1994; Greenberg et al., 1994). All these mutants show some spontaneous necrotic lesion formation in their leaves, increased SA levels, accumulation of MARNA for the SAR gene and markedly increased disease resistance. At least seven different Isd variants were isolated and characterized (Dietrich et al., 1994; Weymann et.
al., 1995). Classes of mutant strains other interest, cim (c onstitutive i mmunity)
It is a mutant (Lawton et al., 1993). See also U.S. Patent No. 5,792,904 and International PCT Application WO 94/16077. Like the Isd mutants and acd2, the cim mutants have elevated levels of SA and SAR gene expression and resistance, but in contrast to Isd or acd2, detectable damage to their leaves Did not show. cpr1 ( c onstitutiv
e expresser of PR genes) may be one type of cim mutant; however, the presence of damage on cprl leaves that is not visible under the microscope has been eliminated, so cprl is a variant of the Isd mutant. It would be one type (Bowling et al., 1994).

[0008] Mutants that are blocked in SAR signaling have also been isolated. ndrl (n o
n-race-specific d isease r esistance) is, Pseudomon include various non-toxic genes
It is a mutant that grows both as syringe and Peronospora parasitica containing normally non-toxic genes (Century et al., 1995). Clearly, this mutant is blocked early on SAR signaling. nprl (nonexpresser of PR genes) is INA
A mutant that cannot induce expression of the SAR signaling pathway after treatment (
Cao et al., 1994). eds (e nhanced d isease s usceptibility ) mutants after inoculation of low bacterial concentrations were isolated based on the ability to retain the bacterial infection
(Glazebrook et al., 1996; Parker et al., 1996). Certain eds mutants are phenotypically similar to nprl, and recently eds5 and eds53 have been shown to be alleles of nprl (Glazebrook et al., 1996). niml (n oninducible im munity) are, P. parasitica after INA treatment (i.e., those causing cotton powdery mildew) holds proliferation (Delaney et al, 1995;. US Patent No. 5,792,904) is a variant. nim 1
Can accumulate SA after pathogen infection, but cannot induce SAR gene expression or disease resistance. This mutant suggests that it blocks the SA pathway downstream.
nim 1 was impaired in its ability to respond to INA or BTH, suggesting that the above-described block exists downstream of the action of these chemicals (Delaney
et al., 1995; Lawton et al., 1996).

[0009] The Arabidopsis allele has been isolated and characterized, and the mutant has
It was responsible for each of the phenotypes of nim 1 and nprl (Ryals et al, 1997; Cao et al
., 1997). The wild-type NIM1 gene product is a gene in SAR and Arabidopsis
Involved in signal transduction cascades that lead to both vs. genetic disease resistance (Ryals et al., 1997). Ryals et al. (1997) also report the isolation of five more alleles of nim1. These alleles are chemically induced PR-1
It shows a weakly impaired phenotype and a very strongly blocked phenotype in gene expression and fungal resistance. Transduction of the wild-type NPR1 gene into nprl mutants not only complements the mutation that restores the response of SAR induction for PR gene expression and disease resistance, but also results in P. siringe To make them more resistant to infection by C. et al. (Cao et al., 1997). WO 98/06748 describes NPR1 isolation from Arabidopsis and homologs from Nicotiana glutinosa. WO 97/4
9822, WO 98/26082 and WO 98/29537.

[0010] Despite the many studies and uses of sophisticated and powerful cereal defense methods, including genetic transformation of plants, disease losses amount to billions of dollars annually. Therefore, there is a need to develop new grain protection methods based on the accumulated understanding of the genetic mechanisms for disease resistance in plants. In particular, there is a need for the identification, isolation and characterization of Arabidopsis NIM1 gene homologs from additional plant species.

In describing the present invention, the following terms will be used, with the definitions as set forth below.

Relevant / functionally linked: The two DNA sequences are physically or functionally related. For example, a promoter or regulatory DNA sequence may encode an RNA or protein if the two sequences are operably linked or are located such that the regulatory DNA sequence affects the level of expression of the encoded or structural DNA sequence. DNA sequence that is "related to"

Chimeric gene: A recombinant DNA sequence in which a promoter or regulatory DNA sequence is operably linked to or associated with a DNA sequence encoding mRNA or expressed as a protein. For example, a regulatory DNA sequence can regulate the transcription or expression of a related DNA sequence.
The regulatory DNA sequence of the chimeric gene usually does not operably bind to related DNA sequences found in nature.

[0014] Coding sequence: a nucleic acid sequence that is transcribed into RNA, such as mRNA, rRNA, tRNA, snRNA
, Sense RNA or antisense RNA. Then, preferably, the RNA is translated in the organism to produce the protein.

Complementarity: Refers to two nucleic acid sequences, including antiparallel nucleic acid sequences, that can pair with each other for the formation of hydrogen bonds between complementary bases in the antiparallel nucleic acid sequences.

Expression: Refers to the transcription and / or translation of a foreign gene or transgene in a plant. In the case of an antisense construct, for example, the expression is
Could only mean translation.

Expression cassette: A nucleic acid sequence capable of directing the expression of a particular nucleotide sequence in a suitable host cell, comprising a promoter operably linked to the nucleotide sequence of interest operably linked to a stop signal. Usually, this will include the sequences required for proper translation of the nucleotide sequence. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. Expression cassette is one of them
It is obtained in a naturally occurring but useful recombinant for heterologous expression. Usually, however, the expression cassette is heterologous with respect to the host,
That is, the particular nucleic acid sequence of the expression cassette is not naturally present in the host cell and must have been introduced into the host cell or ancestor of the host cell upon transformation. Expression of the nucleotide sequence in the expression cassette can be under the control of a constitutive or inducible promoter that initiates transcription only when the host cell is exposed to a particular exogenous stimulus. In the case of a multicellular organism, such as a plant, the promoter may be specific for a particular tissue or organ or growth stage.

Gene: a defined region located in the genome, in addition to the above-described encoding nucleic acid sequence, another defined region containing an inherently regulated nucleic acid sequence responsible for controlling expression, a so-called encoding portion. Transcription and translation regions. One gene may contain additional 5 'and 3' untranslated sequences and termination sequences. Additional sequences that may be present (
Element) is, for example, an intron.

Heterologous DNA sequence: As used herein, “heterologous DNA sequence”, “exogenous DNA”
The term "segment" or "heterologous nucleic acid" refers to a sequence that originates from a foreign source to a particular host cell, or, if from the same source, has been modified with its original ability, respectively. That is, heterologous genes in a host cell include genes that are endogenous to the particular host cell, but are modified, for example, by the use of DNA shuffling. The term also includes non-naturally occurring complex copies of the naturally occurring DNA sequence. Thus, the term refers to a DNA segment located within a host cell nucleic acid that is foreign or heterologous to the cell or that is homologous to the cell but not normally found. The exogenous DNA segment is expressed, resulting in the exogenous polypeptide.

Homologous DNA sequence: A DNA sequence naturally related to the host cell into which it is introduced.

Isocording: When a nucleic acid sequence encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by the reference nucleic acid sequence,
The nucleic acid sequence is isocoded with the reference nucleic acid sequence.

Isolated: In the context of the present invention, an isolated nucleic acid molecule or an isolated enzyme is a nucleic acid that exists free of its natural environment by the human hand and is therefore not a natural product. A molecule or an enzyme. An isolated nucleic acid molecule or enzyme may exist in a purified form, or may exist in a non-native environment, eg, a recombinant host cell.

Minimal promoter: A promoter sequence, particularly a TATA sequence, which is inactive or has a greatly reduced promoter activity in the absence of upstream activation.

Native: means a trait gene present in the genome of a non-transformed cell.

Natural Origin (naturally occurring): The term “natural origin (naturally occurring)” describes a substance that can be found in nature as distinct from being artificially produced by humans. Used to do. For example, a protein or nucleotide sequence that can be isolated from a natural source and that is present in an organism (including viruses) that has not been artificially modified by humans in the laboratory is a natural source.

NIM1: a gene published by Ryals et al., (1997), which
Participates in the AR signal transduction cascade.

NIM1: a protein encoded by the NIM1 gene.

Nucleic acid: The term “nucleic acid” is in either a single or double helix,
1 shows deoxynucleotides or ribonucleotides and polymers thereof. Unless specifically limited, the term includes nucleic acids that have similar binding properties to a reference nucleic acid, and that include known analogs of naturally occurring nucleotides that are metabolized in a manner similar to the naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence inherently includes conservatively modified variants thereof (eg, degenerate codon substitutions) and complementary sequences, as well as the sequences explicitly set forth. Specifically, substitution of degenerate codons is achieved by creating a sequence in which the third position of one or more selected codons is replaced with mixed residues and / or deoxyinosine residues. (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol.
Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8: 91-98.
(1994)). The terms “nucleic acid” or “nucleic acid sequence” may be used interchangeably with gene, cDNA and mRNA encoded by the gene. As used herein, a nucleic acid molecule is preferably a segment of DNA. Nucleotides are indicated by their bases by the following standard abbreviations: adenine (A), cytosine (C), thymine
(T) and guanine (G).

ORF: reading frame Plant: whole plant

Plant cell: The structural and physiological unit of a plant consisting of protoplasts and cell walls. Plant cells can be in the form of isolated single cells or cultured cells or in the form of highly organized units, such as plant tissues, plant organs or whole plant units.

Plant cell culture: The culture of plant units, such as protoplasts, cell culture cells, cells in plant tissue, pollen, pollen tubes, ovules, embryo sac, zygotes and embryos at various stages of development.

Plant sample: leaf, stem, root, flower or part of flower, fruit, pollen, egg cell, zygote,
Seed, cutting, cell or tissue culture or other part or product of a plant.

Plant Organs: Clear, visually constructed and differentiated parts of plants, such as roots, stems,
Leaves, buds or embryos.

Plant tissue: A group of plant cells organized into structural and functional units. Includes any plant tissue in a plant or in culture. The term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue cultures and all groups of plant cells organized in structural and / or functional units. Use of this term is not intended to exclude all specific types of the above plant tissue, or to exclude other plant tissue types other than those encompassed by this definition, or in the absence of this definition. .

Promoter: An untranslated DNA sequence site upstream of the coding region that contains the binding site for RNA polymerase II and initiates transcription of DNA. The promoter region may include additional sequences that act as regulatory sequences for gene expression.

Protoplasts: an isolated plant cell that has no or only part of the cell wall

Purified: The term “purified”, when applied to a nucleic acid or protein, causes the nucleic acid or protein to essentially separate another cellular component that is naturally related. Indicates that it is not included. It can be in either dry or aqueous form, but is preferably in a homogeneous state. Purity and homogeneity are usually measured using analytical chemistry techniques, such as polyacrylamide gels or high performance chromatography. The major type of protein present in the preparation is substantially purified. Protein is indicated by the occurrence of a major band in gel electrophoresis. In particular, it means that the nucleic acid or protein is at least about 50% pure, more preferably at least about 85% pure, and most preferably at least about 99% pure.

Recombinant DNA molecule: A combination of DNA molecules linked together using recombinant DNA technology.

Regulatory sequence: A sequence involved in controlling the expression of a nucleotide sequence. Regulatory sequences, including promoters, are operably linked to the nucleotide sequence of interest and the stop signal. Usually, they also include sequences necessary for proper translation of the nucleotide sequence.

Selectable marker genes: Genes expressed in plant cells confer selective advantages on the cells. The selective advantage retained in cells transformed with a selectable marker gene is a negative selection drug, compared to the growth of untransformed cells,
For example, it may be present because it can grow in the presence of antibiotics or herbicides. The selective advantage retained by the transformed cells is due to enhanced or novel permissiveness utilizing added compounds as nutrients, growth factors or energy sources, compared to the growth of untransformed cells. Will. Selectable marker gene refers to a gene or combination of genes whose expression in a plant cell confers on the cell both selective and positive advantages.

Significant increase: An increase in enzyme activity above the inherent error limit in the assay technique, preferably about twice or more than the increase or the wild-type enzyme activity in the presence of the inhibitor, more preferably About a 5-fold or more, most preferably about a 10-fold or more increase.

In the context of two or more nucleic acid or protein sequences, the term “identical” or “percent identity” is the same or greater than the maximum as determined using one of the following sequence comparison algorithms or visual assays. When considered a match, it refers to two or more sequences or subsequences having a particular percentage of identical amino acids or nucleic acids.

Substantially identical: In two nucleic acid or protein sequences, the term “substantially identical” is measured and compared using one of the following sequence comparison algorithms or visual assays to determine a maximum match. When compared and aligned, it indicates two or more sequences or subsequences that have at least 60%, preferably 80%, more preferably 90-95%, and most preferably at least 99%, nucleic acid or amino acid residue identity. Preferably, the substantial identity is at least about 50 residues in length, more preferably at least 100 or more residues, most preferably at least about 100 or more residues of the sequence. And the most preferred sequences are substantially identical, of about 150 residues or more. In a most preferred embodiment, the sequences are substantially identical over the full length of the coding sequence. Further, substantially identical nucleic acid or protein sequences have substantially the same function.

For sequence comparison, usually one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are:
Entered into the computer. Represents the lower array coordinate axis, if necessary, and indicates the algorithm program parameters of the array. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence, based on the program parameters provided.

[0045] Optimal matches for sequences for comparison can be found, for example, in Smith & Waterman, Adv. Appl.
Math. 2: 482 (1981), the local homology algorithm, or Needleman & Wunsch,
J. Mol. Biol. 48: 443 (1970), by the search for similarity algorithm in Pearson & Lipman,
Nat'l. Acad. Sci. USA 85: 2444 (1988). Searching for similar methods, these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetic
s Software Package, Genetics Computer Group, 575 Science Dr., Madison, W
This can be done by computerized means of I) or by visual inspection (see generally Ausubel et al., Infra).

One example of an algorithm that is suitable for determining% sequence identity and sequence homology is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:
403-410 (1990). Software for performing BLAST analysis is available at the National Center for Biotechnology Information (http: //www.ncbi.nim.nih
.gov /).

The algorithm uses a length W in the suspected sequence that matches or satisfies the threshold score T for some positive values when aligned with words of the same length in the database sequence. By identifying the short words of, the first identified high score sequence pairs (HSPs) are included. T is shown as the neighborhood word score limit (Altschul et al., 1990). These starting word hits act as seeds for initiating searches to find longer HSPs containing them.
Subsequently, this word hit expands in each sequence according to both searches, as long as the cumulative sequence hit can increase the cumulative sequence score. The cumulative score is calculated using the nucleotide sequence and using the parameters M (reward score for a set of matching residues; always greater than or equal to 0) and N (penalty score for mismatching residues; always less than or equal to 0). Calculated. For amino acid sequences, a cumulative score was calculated using a matrix to be scored. The stoppage of word hit growth in each direction is due to the cumulative sequence score decreasing to the quantity X from its highest achieved value, with a cumulative score of 0 or less due to a negative score residue sequence of 1 or more. Or reaches the end of any sequence. The BLAT algorithm parameters W, T and X measure the sensitivity and speed of the sequence. BLASTN program (
Nucleotide sequence) uses the word length (W), expected value (E) of 10, cutoff of 100, M = 5, N = -4, and a comparison of both strands as default. For amino acid sequences, the BLASTP program calculates the word length (W) 3, expected value (E) 10, BLO
SUM62 score matrix (Henikoff & Henikoff, Proc. Natl. Acad. Sci. U
SA 89: 10915 (1989)) used as default.

In addition to measuring% sequence homology, the BLAST algorithm performed a similar statistical analysis between the two sequences (eg, Karlin & Altschul, Proc. Nat'l. A
cad. Sci. USA 90: 5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the probability of least sum (P (N)), which is an indicator of the probability that a match between two nucleic acid or amino acid sequences will occur by chance. For example, a test nucleic acid may have a minimum total probability of about a comparison between a test nucleic acid sequence and a reference nucleic acid sequence.
If it is less than 0.1, preferably less than about 0.01, and most preferably less than about 0.001, it is considered similar to the reference sequence.

Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase "hybridizes specifically to" refers to a sequence that, when present in a complex mixture (eg, whole cell) DNA or RNA, binds the molecule to only a particular nucleic acid sequence under stringent conditions. , Indicates duplication or hybridization. "Substantial binding" refers to complementary hybridization between a probe nucleic acid and a target nucleic acid, which can reduce the stringency of the hybridization medium to achieve the detection of the desired target nucleic acid sequence and can accommodate a small amount. Inconsistencies.

In nucleic acid hybridization experiments, such as Southern and Northern hybridizations, “stringent hybridization conditions” and “stringent hybridization wash conditions” are different for different environmental parameters. Long sequences hybridize specifically at elevated temperatures. Detailed guidelines for nucleic acid hybridization can be found in Tijssen (1993) Laboratory Techniques in Biochemistry an.
d Molecular Biology-hybridization with nucleic acid Probes Part1 chapter
2 "Overview of principles of hybridization and the strategy of nucleic acid a
cid probe assays "in Elsevier, New York. Generally, high stringency hybridization and washing conditions are below the thermal melting point (Tm) for a particular sequence at a defined ionic strength and pH. A choice of about 5 ° C. Usually, under “stringent conditions,” a probe will hybridize to its target sequence but not to another sequence.

Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are chosen equal to Tm for a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids with more than 100 complementary residues on a filter in a Southern or Northern blot is at 42 ° C., 50% formamide with 1 mg of heparin. Hybridization is performed overnight. An example of high stringency wash conditions is 0.15 M NaCl at 72 ° C. for about 15 minutes. An example of a stringent wash condition is a 0.2 × SSC wash at 65 ° C. for 15 minutes (see Sambroo for a description of SSC buffer).
k, infra). High stringency washes are often performed with low stringency washes to remove background probe signals. For example, highly stringent washes of the example medium for duplication of 100 or more nucleotides at 45 ° C. with 1 × SSC
Minutes. A low stringency wash of the example medium for an overlap of, for example, 100 or more nucleotides is 4-6 × SSC at 40 ° C. for 15 minutes. Short probes (for example, about 1
For 0 to 50 nucleotides), stringent conditions generally include a salt concentration of about 1.0 M Na ion or less, usually at a pH of 7.0 to 8.3 and about 0.01 to 1.0 M Na ion concentration (or another Salt) and the temperature is usually at least about 30 ° C. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. In general,
A signal with a noise ratio of more than two-fold (or more) observed for an unrelated probe in a particular hybridization assay indicates detection of specific hybridization. Nucleic acids that do not hybridize to each other under stringent conditions are substantially identical if the proteins that encode them are substantially identical. This occurs, for example, when a copy of a nucleic acid is created (constructed) using the maximum codon degeneracy permitted by the genetic code.

The following is an example of a set of hybridization / wash conditions that can be used to clone homologous nucleotide sequences that are substantially identical to a reference nucleotide sequence of the present invention. Reference nucleotide sequence, washing with 1 mM EDTA 2X SSC, at 50 ° C., 7% sodium dodecyl sulfate (SDS), in 0.5 M NaP0 _4, more preferably 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0 ₄ , 1 mM EDTA,
Preferably, it hybridizes to a reference nucleic acid sequence. 0.1 while washing with 0.5 × SSC
% SDS, washed at 50 ° C, more preferably 7% sodium dodecyl sulfate (SDS), 0.5%
In M NaP0 _4, 1 mM EDTA, at 50 ° C., washing with 0.5X SSC, preferably, 50 ° C.
In washing with 0.1 X SSC, 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0 ₄
, 1 mM EDTA. 0.1% by 50 ° C. SDS, more preferably, at 50 ° C., 7% sodium dodecyl sulfate _{(SDS), 0.5 M NaP0 4} , 1 mM EDTA, 65 0.1 X SSC at ° C., 0.1% SDS
Wash with.

Another indication that two nucleic acid sequences or proteins are substantially identical is that the protein encoded by the first nucleic acid may immunologically cross-react or specifically react with the protein encoded by the second nucleic acid. It is what combines. That is, a protein is usually substantially identical to a second protein, for example, when the two proteins differ only by conservative substitutions.

The terms “specifically (or selectively) binds to an antibody” or “specifically (or selectively) immunoreactive” when referring to a protein or peptide, refer to protein and other A binding reaction that determines the presence of a protein in the presence of a heterogeneous population of biologics. That is, under established immunoassay conditions, a particular antibody binds to a particular protein and does not bind to other proteins in the sample in significant amounts. Under such conditions, specific binding to the antibody may require the specifically selected antibody for a particular protein. For example, an antibody raised from a protein having an amino acid sequence encoded by any of the nucleic acid sequences of the present invention may be an antibody that specifically immunoreacts with a protein and does not specifically immunoreact with another protein except for a polymorphic variant. May be selected to obtain A variety of immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein. For example, solid phase ELISA immunoassays, Western
Blots or immunohistochemistry are commonly used to select monoclonal antibodies that react specifically with the protein. For a description of immunoassay formats and conditions that can be used to measure specific immune activity, see (Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Ha
rbor Publications, New York "Harlow and Lane"). Usually, a specific or selective reaction is at least two times background signal or noise and more usually more than 10 to 100 times background.

A “conservatively modified mutation” of a particular nucleic acid sequence is a nucleic acid sequence that encodes an identical or substantially identical amino acid sequence, or a nucleic acid that does not encode an amino acid sequence, for a substantially identical sequence. Shows the sequence. Because of the degeneracy of the genetic code, many functionally identical nucleic acids encode all of the resulting polypeptides. For example, the codons CGT, CGC, CGA, CGG, AGA and AGG all encode the amino acid arginine. Thus, at every position where arginine is specified by a codon, this codon can be changed to any of the corresponding codons described without changing the encoded protein. Such a nucleic acid mutation is a “silent mutation”, which is a type of “conservatively modified mutation”. Every nucleic acid sequence described in the specification which encodes a protein describes all possible silent variations, unless otherwise specified. As will be appreciated by those skilled in the art, modifying each codon in a nucleic acid (except for ATG, which is usually the only codon for methionine) to produce functionally identical molecules by standard techniques. Can be.
Thus, each "silent mutation" of a nucleic acid which encodes a protein is implicit in each described sequence.

In addition, one technique involves changing, adding, or deleting one amino acid or a low percentage of amino acids in a coding sequence (usually less than 5%, more typically less than 1%). The deletion or addition of each substituent that is will be recognized as a "conservatively modified mutation" if the change results in a substitution of the amino acid by a chemically similar amino acid. Conservative substitution tables indicating functionally similar amino acids are known to those skilled in the art. The following five groups include amino acids having conservative substitutions with each other: aliphatic; glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I); aromatic; phenylalanine (F ), Tyrosine (T), tryptophan (W
); Sulfur-containing; methionine (M), cysteine (S); basic; arginine (A)
, Leucine (K), histidine (H); acidic; including aspartic acid (D), glutamic acid (E), asparagine (N), and glutamine (Q). Creighton (1984) Prot
See ein, WH Freeman and Company. In addition, each substitution, deletion or addition that changes, adds or deletes one amino acid or a low percentage of amino acids in a coding sequence is also a "silent mutation."

A “subsequence” refers to a nucleic acid or amino acid sequence that includes a portion of a longer nucleic acid or amino acid (eg, protein) sequence, respectively.

[0058] A nucleic acid is used when additional nucleotides (another similar molecule) are incorporated into the nucleic acid.
"Extend". Most commonly, this is done by a polymerase (eg, a DNA polymerase), for example, a polymerase that adds a sequence at the 3 ′ end of a nucleic acid.

[0059] Two nucleic acids "rejoin" when sequences from each two nucleic acids are combined in a progeny nucleic acid. Two sequences recombine "directly" if both nucleic acids are substrates for recombination. Two sequences are "indirect recombination" if the sequences recombine with an intermediary such as a cross-over oligonucleotide. For indirect recombination, one sequence is the actual substrate for recombination, and in some cases, the sequence is not a substrate for recombination.

A “specific binding affinity” between two molecules, eg, a ligand and a receptor, refers to preferential binding of another molecule in a mixture of molecules. The binding of molecules is
When the binding affinity is about 1 × 10 ⁴ M ⁻¹ to about 1 × 10 ⁴ M ⁻¹ or more,
Can be considered specific.

Transformation; a method for introducing heterologous DNA into a host cell or organism.

The terms “transform”, “transformation” and “recombinant” refer to a host organism, such as a bacterium or a plant, into which the heterologous DNA has been introduced. The nucleic acid molecule can be stably introduced into the host genome, or the nucleic acid molecule can be present as an extrachromosomal molecule. Such extrachromosomal molecules may be self-replicating. Transformed cells, tissues or plants are understood to include not only the end products of the transformation process, but also their transformed progeny. "Untransformed", "untransformed" or "
A “non-recombinant” host refers to a wild-type organism that does not contain a heterologous nucleic acid molecule, such as a bacterium or a plant.

The present invention solves the above need by providing Arabidopsis NIM1 gene homologs from additional species of plants. In particular, the present invention relates to Nicotiana tabacum (NIM1 gene encoding a protein believed to be involved in a signal transduction cascade that responds to biological and chemical inducers that lead to systemically acquired resistance in plants). Tobacco), Lycopersicon esculentum (tomato), Brassica napus (fat seed), Arabidopsis thaliana, Beta vulgaris
(Sugar beet), Helianthus annuus (sunflower) and Solan
um tuberosum (Poto)

To that end, the present invention provides SEQ ID NOs: 2, 4, 6, 8, 16, 18, 20, 30, 32, 34, 36
, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 62, 64, 66, 68, 70, 72 or 74 directed to an isolated nucleic acid molecule. I do.

In another embodiment, the present invention provides a method of SEQ ID NOS: 1, 3, 5, 7, 15, 17, 19, 29, 31,
33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 61, 63, 65, 67, 69
, 71 or 73 is an isolated nucleic acid molecule.

In a further embodiment, the present invention provides SEQ ID NOs: 1, 3, 5, 7, 15, 17, 19, 29
, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 61, 63, 65, 67
, 69, 71 or 73 consecutive at least 20, 25, 30, 35, 40, 45 or 50 (preferably 20) base pair portions and at least 20, 25, 30, 35, 4
It is directed to an isolated nucleic acid molecule containing a nucleotide sequence comprising 0, 45 or 50 (preferably 20) contiguous base pair portions.

In another embodiment, the present invention provides a method comprising:
24, SEQ ID NOs: 22 and 24, SEQ ID NOs: 25 and 28, SEQ ID NOs: 26 and 28 or amplified from a Lycopersicon esculentum DNA library using the polymerase chain reaction with a set of primers as set forth in SEQ ID NOs: 59 and 60 Directed to an isolated nucleic acid molecule comprising a possible nucleotide sequence.

In yet another embodiment, the present invention provides a method comprising SEQ ID NO: 22 and 24 or SEQ ID NO:
Be using the polymerase chain reaction with a set of primers described in 26 and 28.
directed to an isolated nucleic acid molecule comprising a nucleotide sequence that can be amplified from a T. vulgaris DNA library.

In a further embodiment, the present invention is directed to an isolated nucleic acid molecule comprising a nucleotide sequence that can be amplified from Helianthus annuus DNA using the polymerase chain reaction with a set of primers of SEQ ID NOs: 26 and 28.

In another embodiment, the invention relates to SEQ ID NOs: 21 and 24, SEQ ID NOs: 21 and 23
, SEQ ID NOs: 22 and 24, SEQ ID NOs: 25 and 28, or SEQ ID NOs: 26 and
Solanum tuberosum using polymerase chain reaction with 28 sets of primers
It is directed to an isolated nucleic acid molecule containing a nucleotide sequence that can be amplified from a DNA library.

In a further embodiment, the invention relates to SEQ ID NOS: 9 and 10 or SEQ ID NO: 26
Brassica nap using polymerase chain reaction with a set of primers and 28
directed to an isolated nucleic acid molecule containing a nucleotide sequence that can be amplified from a us DNA library.

In another embodiment, the present invention relates to the described SEQ ID NOs: 13 and 14, SEQ ID NO: 21
And 24, or SEQ ID NOs: 22 and 24, directed to an isolated nucleic acid molecule comprising a nucleotide sequence that can be amplified from an Arabidopsis thaliana DNA library using the polymerase chain reaction.

In a further embodiment, the invention relates to SEQ ID NOS: 9 and 10, SEQ ID NOS: 11 and 12, SEQ ID NOs: 21 and 24, SEQ ID NOs: 22 and 24, SEQ ID NOs: 25 and 28
, Or an isolated nucleic acid molecule comprising a nucleotide sequence that can be amplified from a Nicotiana tabacum DNA library using the polymerase chain reaction with the set of primers set forth in SEQ ID NOs: 26 and 28.

In a further embodiment, SEQ ID NOs: 1, 3, 5, 7, 15, 17, 19, 29, 31, 33, 35
, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 61, 63, 65, 67, 69, 71 or 73 with the first 20 nucleotides of the coding sequence (CDS) A polymerase chain reaction with a set of primers containing the reverse complement of the last 20 nucleotides is used to direct an isolated nucleic acid molecule containing a nucleotide sequence that can be amplified from a plant DNA library.

The present invention relates to a chimeric gene comprising a promoter active in plants operably linked to the coding sequence of the NIM1 homologue of the present invention, a recombinant vector comprising such a chimeric gene, and a host cell. And a stable host transformed with such a vector. Preferably, the host is one of the following agriculturally important cereal plants: rice, wheat, barley, rye, canola, sugar corn, corn, potato, carrot, sweet potato, sugar beet, sugar bean , Peas, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, pumpkin, pepo pumpkin, cucumber, apple, pear, quince, plum, cherry, peach , Nectarines, apricots, strawberries, grapes, raspberries, blackberries, pineapples, avocados, papayas, mangos, bananas, soybeans, tobacco, tomatoes, sorghum and sugarcane. The present invention also includes species derived from the plant of the present invention.

Further, the present invention provides a method for binding functionally to the coding sequence of a NIM 1 homolog of the invention.
A method for increasing SAR gene expression in plants by expressing a chimeric gene containing the promoter activity itself in the plant is directed. The encoded protein is expressed at higher levels in transformed plants than in wild-type plants.

Further, the present invention provides a method for binding functionally to the coding sequence of a NIM 1 homologue of the present invention.
The present invention is directed to a method for maintaining disease resistance in plants by expressing a chimeric gene containing a promoter activity in the plant itself. The encoded protein is expressed at higher levels in transformed plants than in wild-type plants.

Further, the present invention is directed to a PCR primer selected from the group consisting of SEQ ID NOs: 9-14, 21-28, 59 and 60.

The present invention provides SEQ ID NOS: 1, 3, 5, 7, 15, 17, 19, 29, 31, 33, 35, 37, 39, 41
, 43, 45, 47, 49, 51, 53, 55, 57, 61, 63, 65, 67, 69, 71 or 73 the first 20 nucleotides and the reverse of the last 20 nucleotides of the coding sequence (CDS) A set of primers corresponding to the complement, or SEQ ID NOS: 9 and 10, SEQ ID NOS: 11 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 21 and 2
4, SEQ ID NOS: 22 and 24, SEQ ID NOs: 21 and 23, SEQ ID NOs: 25 and 28,
A signal transducer that can lead to tissue resistance in plants, comprising amplifying from a Nicotiana tabacum DNA library using the polymerase chain reaction with a set of primers set forth as SEQ ID NOs: 26 and 28, or SEQ ID NOs: 59 and 60 -A method for isolating NIM1 homologues for the duplication cascade.
In a preferred embodiment, the plant DNA library is Nicotiana tabacum (tobacco)
, Lycopersicon esculentum (tomato), Brassica napus (oilseed), Arabidop
sis thaliana, Beta vulgaris (sugar cane), Helianthus annuus (sunflower)
Or Solanum tuberosum (pot).

[0080] Northern data for some of the NIM1 homologs described herein showed constitutive expression or BTH inducibility. Homologs of the described NIM1 gene suggest that it encodes a protein involved in a signal transduction cascade in response to biological and chemical inducers, which was systematically acquired in plants. Shows resistance. The present invention also relates to the transformable expression of such NIM1 homologues in plants to enhance SAR gene expression and enhance disease resistance.

The DNA sequences of the present invention may be isolated using the techniques described in the Examples below, or by PCR using the sequences set forth as bases for constructing PCR primers. For example, an oligonucleotide having the first and last about 20-25 contiguous nucleotides of SEQ ID NO: 7 (e.g., nucleotides 1-20, 1742-1761 of SEQ ID NO: 7) is a source plant (Arabidopsis thaliana) Can be used as PCR primers to amplify a cDNA sequence (SEQ ID NO: 7) directly from a cDNA library from S.I. Another DNA sequence of the present invention can also be amplified by PCR from a cDNA or genomic DNA library of each plant using the ends of the DNA listed in the sequences listed as bases for PCR primers. .

It is believed that the transformed expression of the NIM1 homologs of the present invention in plants results in broad immunity to plant pathogens. This pathogen is not limited to a virus or pathogen, for example, tobacco or cucumber mosaic virus, ring spot virus or necrosis virus, pelagonium cigar virus, red clover mottle virus, tomato bushy stunt virus,
Other viruses; fungi, such as oomycetes, such as Phythophthora parasitica
And Peronospora tabacina; bacteria such as Pseudomonas syringe and Ps
Insects such as eudomonas tabac; aphids, such as Myzus persicae; and lepidoptera, such as Heliothus spp; and nematodes, such as Meloidogyne incognita. The vectors and methods of the present invention are not limited to fluffy powdery mildew, but include legume plants such as Scleropthora macrospora, Sclerophthora rayissiae, Sclerospor
a graminicola, Peronosclerospora sorghi, Peronosclerospora philippinens
is, Peronosclerospora sacchari and Peronosclerospora maydis; rusts suc
h as Puccinia sorphi, Puccinia polysora and Physopella zeae; other fungi,
For example, Cercospora zeae-maydis, Colletotrichum graminicola, Fusarium mono
liforme, Gibberella zeae, Exserohilum turcicum, Kabatiellu zeae, Ery
siphe graminis, Septoria and Bipolaris maydis; and bacteria such as Er
Useful for many plant diseases, including winia stewartai.

The methods of the present invention can be used to confer disease resistance on a variety of plants, including gymnosperms, monocots and dicots. However, disease resistance can be conferred on all plants within these broad classes, but especially on agriculturally important cereal plants such as rice, wheat, barley, rye, rice, wheat, barley, rye. , Oilseed, corn, potato, carrot, sweet potato, sugar beet, sugar beet, haricot bean, peas, chicory, lettuce, cabbage, cauliflower, broccoli, cabbage, radish, spinach, asparagus, onion, garlic, eggplant, pepper, Celery, carrot, pumpkin, pepo pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, Soybean, Baco useful tomato, in plants such as sorghum acids and sugar cane.

A NIM1 homologue encoding a sequence of the present invention can be introduced into an expression cassette designed for plants using standard genetic engineering techniques to create a chimeric gene of the present invention. Specific regulatory sequences, such as promoters, signal sequences,
The choice of 5 'and 3' untranslated sequences and appropriate enhancers to achieve the desired pattern and expression level in the chosen plant host is within the skill of the art. The resulting molecule containing the individual sequences linked in the appropriate reading frame may be introduced into a vector that can be transformed in a host plant cell.

Examples of promoters that can function in plants or plant cells (ie, those that can drive expression of the relevant coding sequence, eg, encode for NIM1 homologs in plant cells) are the Arabidopsis and corn ubiquitin promoters ; Cauliflower mosaic virus (CaMV) 19S or 35S promoter and CaMV double promoter; rice actin promoter; tobacco, arabidopsis, or maize-derived PR-1 promoter; opaline synthase promoter; ribulose bisphosphate carboxylase (ssuRUBISCO) And other promoters. Particularly preferred is the Arabidopsis ubiquitin promoter. The promoter, itself, can be modified to manipulate promoter strength, to increase expression of the relevant coding sequence associated with art-recognized methods. Preferred promoters for use in the present invention are those that confer high levels of constitutive expression.

A signal peptide or transit peptide may be fused to a NIM1 homolog coding sequence in a chimeric DNA construct of the present invention to direct the transfer of an expressed protein to a desired site of action. Examples of signal peptides include those naturally associated with plant pathogenic gene-related proteins such as PR-1, PR-2 and the like (
See, for example, Payne et al., 1988). Transit peptides are chloroplast transit peptides, e.g., Von Heijne et al (1991), Mazur et al.
1987) and Vorst et al. (1988); and mitochondrial transit peptides, such as those described in Boutry et al. (1987). Also included are sequences that effect localization of the encoded protein to various cellular compartments, such as the vacuole. See, for example, Neuhaus et al. (1991) and Chrispeels (1991).

The chimeric DNA constructs of the present invention may include multiple promoter copies or multiple NIM1 homolog coding sequences of the present invention. In addition, the constructs can include the coding sequence for the marker, and the coding sequence for each other peptide, such as a signal peptide or transit peptide, in the appropriate reading frame with another functional sequence in the DNA molecule. Preparation of such constructs is within the ordinary skill in the art.

Useful markers include peptides that provide resistance to herbicides, antibiotics or drugs. For example, protoporphyrinogen oxidase (protoporphyrinog)
en) Inhibitors, hygromycin, kanamycin, G418, gentamicin, linocomycin, methotrexate, glyphosate, phosphinothricin (phosphino
thricin) or analogs thereof. These markers can be used to select cells transformed with the chimeric DNA constructs of the present invention from untransformed cells. Other useful markers are peptidic enzymes that can be easily detected by a visual reaction, such as a color reaction, such as luciferase, β-glucuronidase or β-galactosidase.

A chimeric gene constructed for expression in a plant as described herein can be introduced into plant cells in a number of art-recognized ways. As those skilled in the art are aware, the choice of method depends on the type of plant targeted for transformation (i.e., monocotyledonous or dicotyledonous) and / or organ (i.e., cell nucleus, chloroplast, mitochondria). Depends on.

A suitable method for transforming plant cells is microinjection (Crossway
et al., 1986), electroporation (Riggs et al., 1986), Agrobacterium-mediated transformation (Hinchee et al., 1988; Ishida et al., 1996), direct gene transformation (Paszkowski et al. ., 1984; Hayashimoto et al., 1990) and Agracetus Inc., Maison, Wisconsin, and Dupont.
ont, Inc., Wilmington, Delaware) ballistic particle acceleration (see, eg, US Patent 4,945,050; and McCabe et al., 1988). Also, Weiss
inger et al. (1988); Sanford et al. (1987) (onion); Christou et au (198
8) (soy); McCabe et al., (1988) (soy); Datt et al. (1990) (rice); Klein
et al., (1988) (maize); Klein et au (1988) (maize); Klein
et al., (1988) (maize); Fromm et al. (1990); and Gordon-Kamm et.
al. (1990) (maize); Svab et al) (1990) (tobacco chloroplast); Go
rdon-Kamm et au (1993) (maize); Shimamoto et al. (1989) (US); Chri
stou et al. (1991) (US); Datta et al. (1990) (US); European Patent Appli
cation EP 0 332 581 (orchardgrass and other Pooideae); Vasil et al. (19
93) (wheat); Weeks et al. (1993) (wheat); Wan et al. (1994) (barley); Jahne
et al. (1994) (barley); Umbeck et al. (1987) (cotton); Casas et al. (1993) (
(Sorghum); Somers et al. (1992) (oats); Torbert et al. (1995) (
Oats); Weeks et al., (1993) (wheat); WO 94/13822 (wheat); and Nehr
a et al) (1994) (wheat). One particularly preferred set of embodiments for introducing recombinant DNA molecules into corn by microinjection bombardment is K
oziel et al. (1993); Hill et al. (1995) and Koziel et al. (1996). An additional preferred embodiment is a method for transforming protoplasts for maize as described in EP 0 292 435.

A chimeric gene containing a coding sequence for a NIM1 homolog can be transformed into a particular plant species and inherited in the same species using conventional growth techniques, or transformed into a homologous mutant, especially a commercially available mutant. Can be propagated. Particularly preferred plants of the present invention include the agriculturally important cereal plants described above. The genetic characteristics of the above-described transformed seeds and those entrapped in plants can be inherited by sexual reproduction and maintained and transmitted to progeny plants.

EXAMPLES The present invention is further described by the following detailed methods, preparations and examples. The examples are provided for illustrative purposes only and are not configured to limit the scope of the invention. Standard recombinant DNA and molecular cloning techniques used herein are known to those of skill in the art and are described in Sambrook, et al., 1989; TJS.
ilhavy, ML Berman and LW Enquis 1984; and Ausubel, FM et al.,
1987 et al.

I. Isolation of homologues of the Arabidopsis NIM1 gene Example 1: Isolation of NIM1 homologues from Nicotiana tabacum Plasmid DNA from large excisions of phage from a tobacco cDNA library
Is used as a template for PCR using the following primer set: 5'-AGATTATTGT
CAAGTCTAATG-3 '(SEQ ID NO: 9) + 5'-TTCCATGTACCTTTGCTTC-3' (SEQ ID NO: 10),
And 5'-GCGGATCCATGGATAATAGTAGG-3 '(SEQ ID NO: 11) + 5'-GCGGATCCTATTTCCTA
AAAGGG-3 '(SEQ ID NO: 12). The cycling conditions are 94 ° C. for 1 minute, 40 ° C. for 1 minute and 1.5 minutes at 72 ° C., and the reaction is preferably carried out for 40 cycles. PCR products
It was run on an agarose gel, excised and cloned with pCRII-TOPO (Invitrogen).

The full length cDNA sequence of the tobacco NIM1 homolog is shown in SEQ ID NO: 1. The protein encoded by this cDNA sequence is shown in SEQ ID NO: 2. Tobacco NIM1 homologues comprising SEQ ID NO: 1 were obtained from NRRL (Agricultural Research Service, Patent Culture
Collection, Northern Regional Research Center, 1815 North University S
treet, Peoria, Illinois 61604, US A), pNOV12 on August 17, 1998.
Deposited as 06 and having accession number NRRL B-30051.

Example 2: Isolation of NIM1 homologue from Lycopersicon esculentum A phagemid was excised from the tomato λZAPII cDNA library using the Stratagene protocol. Phagemids (plasmids) were mass transformed into E. coli XL1-Blue in 10 pools of each of approximately 80,000 clones, and DNA was extracted from these pools. This pool contains the following primers: 5'-AGATTATTGTCAAGTCTAATG-3 '(
Screened by PCR for the presence of NIM1 homologs by PCR with SEQ ID NO: 9) and 5'-TTCCATGTACCTTTGCTTC-3 '(SEQ ID NO: 10).

The sequences amplified from the pool were confirmed to contain NIM1 homologs by cloning and sequencing PCR amplified DNA fragments. The pool was progressively reduced until a single clone containing the homolog was obtained and screened by PCR using the same primers described above for the presence of the NIM1 homolog. Finally, a cDNA clone containing the partial gene with the 5 ′ end deleted was 5′RACE (Rapid Amplification fo
cDNA Ends) was used to obtain the full-length sequence of the gene.

The full length cDNA sequence of the tomato NIM1 homolog is shown in SEQ ID NO: 3, and the protein encoded by this cDNA sequence is shown in SEQ ID NO: 4. The NIM1 homolog of tomato containing SEQ ID NO: 3 was designated as pNOV1204 by NRRL (Agricultural Research Serv.
ice, Patent Culture Collection, Northern Regional Research Center, 181
5 North University Street, Peoria, Illinois 61604, USA), 1998
The deposit was deposited on August 17, 2008, and the deposit number is NRRL B-30050.

Example 3 Isolation of NIM1 Homologues from Brassica napus Phagemids were purified from Brassica napus XZA using the Stratagene protocol.
It was excised from the PllcDNA library. About 80,000 phagemids (plasmids)
E. coli XL1-Blue was mass transformed with 10 pools of each of the clones, and DNA was extracted from these pools. The pool was prepared using the following primers: 5'-AGATTATTGTCAAGTCTAA
Screened by PCR for the presence of NIM1 homologs by PCR with TG-3 ′ (SEQ ID NO: 9) and 5′-TTCCATGTACCTTTGCTTC-3 ′ (SEQ ID NO: 10).

The sequences amplified from the pool were confirmed to contain NIM1 homologs by cloning and sequencing PCR amplified DNA fragments. The pool was progressively reduced until a single clone containing the homolog was obtained and screened by PCR using the same primers described above for the presence of the NIM1 homolog. Finally, in the cDNA clone containing the partial gene with the 5 'end deleted, 5' RACE (Rapid Amplificat
ion fo cDNA Ends) was used to obtain the full-length sequence of the gene.

The partial gene of the NIM1 homologue of Brassica napus is shown in SEQ ID NO: 5,
The protein encoded by the sequence is shown in SEQ ID NO: 6. A Brassica napus NIM1 homolog containing SEQ ID NO: 5 was designated as pNOV1203 by NRRL (Agricultural
Research Service, Patent Culture Collection, Northern Regional Research
Center, 1815 North University Street, Peoria, Illinois 61604, USA) and deposited on August 17, 1998, accession number NRRL B-30049.

Example 4: Isolation of Arabidopsis thaliana NIM1 homologs Examination of BLAST using the Arabidopsis or tomato NIM1 amino acid sequence includes the Arabidopsis genomic sequence from the bacterial artificial chromosome (BAC) F18D8
GenBank entry B26306 was detected. Part of the BAC sequence is predicted to encode a protein (47% amino acid identity) that is significantly similar to NIM1. The following primers
Design for regions of the F18D8 sequence: 5'-TCAAGGCCTTGGATTCAGATG-3 '(SEQ ID NO: 13) and 5'-ATTAACTGCGCTACGTCCGTC-3' (SEQ ID NO: 14).

The primers were used in a PCR reaction with DNA from a pFL61-dependent Arabidopsis cDNA library as a template. Preferred cycling conditions are 94 ° C for 30 seconds, 53 ° C for 30 seconds.
30 seconds at 72 ° C. The reaction is preferably performed for 40 cycles. A PCR product of the expected size (290 base pairs) is detected, and a cDNA clone matching the F18D8 primer is purified from the cDNA library by a continuous purification method. In this method, an increased amount of the library is passed through E. coli and redetected in the presence of clones by PCR. Finally, a single positive clone was obtained and sequenced. The sequence of the clone confirms the presence of an open reading frame with significant homology to NIM1.

The full-length cDNA sequence of the NIM1 homolog of the Arabidopsis thaliana is shown in SEQ ID NO: 7, and the protein encoded by this cDNA sequence is shown in SEQ ID NO: 8.
The Arabidopsis thaliana NIM1 homolog containing SEQ ID NO: 7 is A in E. coli.
tNMLc5, NRRL (Agricultural Research Service, Patent Culture Colle
ction, Northern Regional Research Center, 1815 North University Street
, Peoria, Illinois 61604, USA) and deposited on May 25, 1999, accession number NRRL B30139.

Example 5: Design of degenerate primers The NIM1 gene described above in Example 4 (AtNMLc5-SEQ ID NO: 7) (Ryals et al., 1997)
In addition to the NIM-like gene and Arabidopsis thaliana, three other NIM-like (NM
L) Genome sequence: AtNMLc2 (SEQ ID NO: 15) AtNMLc4-1 (SEQ ID NO: 17) and AtNMLc4
-2 (SEQ ID NO: 19). c [#] indicates the number of chromosomes where a particular NML gene is located. GCG Seqweb multiple sequence alignment program (Pretty, Wiscons
in Genetics Computer Group) using Arabidopsis thaliana (Ryals et al.,
1997), Nicotiana tabacum (Example 1-SEQ ID NO: 1), and Lycopersicon e
NIM1 sequence from sculentum (Example 2-SEQ ID NO: 3), plus Arabidopsis thal
The NML sequences from iana (SEQ ID NOs: 7, 15, 17, and 19) are aligned. Based on this alignment, the three regions yield sufficient conservation to design degenerate PCR primers for PCR amplification of NIM1 homologues from other cereal species, including sunflower, potato, and canola. The primers designed from these conserved regions are shown in Table 1. NIM 1 (AD) primers are available from Arabidopsis thaliana (Ryals et al., 1997), N
The design is performed using only the NIM1 gene derived from icotiana tabacum (Example 1-SEQ ID NO: 1) and Lycopersicon esculentum (Example 2-SEQ ID NO: 3). NIM 2 (A-
D) Primers show four NML sequences from Arabidopsis thaliana (SEQ ID NO: 7, 15, 17, and 19) in addition to these three primers. Primers are Genosy
It is preferably synthesized by Biotechnologies, Inc. (The Woodlands, Texas). The positions of degeneracy are shown in Table 1 by the designation of one or more bases at a single site in the oligonucleotide. "Orientation" indicates whether the primer is oriented in the 3 'end (downstream) or 5' end (upstream) direction of the cDNA.

Table 1: Degenerate primers

[Table 1]

Example 6: PCR amplification of NlM4-like DNA fragments from cereal species NIM-like DNA fragments were obtained from Arabidopsis, tomato, tobacco, sugar beet, sunflower,
Amplification from potatoes and canola using either genomic DNA or cDNA as template. The primer combination used with the expected fragment size
It is described in Table 9 below.

Table 2: Primer combinations and DNA fragment sizes

[Table 2]

The degenerate primer PCR was performed using the GeneAmp PCR System 9700 (PE Applied Biosystems,
Foster City, CA), ready-to-use PCR beads (Amersham, Piscataw
ay, NJ). 20-40 ng genomic DNA or 5-10 ng cDNA, final concentration 0
.8 μM of each primer was used for each reaction. Preferred cycling parameters are: 1 minute at 94 ° C; 3 cycles [30 seconds at 94 ° C; 30 seconds at 37 ° C; 2 minutes at 72 ° C]; 35 cycles [30 seconds at 94 ° C; 30 seconds at 60 ° C. ; 72 ° C for 2 minutes]; 72 ° C for 7 minutes; 4
Keep at ℃. The reaction product is analyzed on a 2% agarose gel and the appropriate size DN
A fragment was cut out. The DNA fragment is, for example, Geneclean III Kit (BIO 101, Inc.,
Isolate from agarose gel using Carlsbad, CA). For example, it was cloned using a TOPO TA cloning kit (Invitrogen Corporation, Carlsbad, CA). Plasmids are available, for example, from the CONCERT Rapid plasmid Miniprep System [Life Technologies, Inc.,
Rockville, MD] and sequenced according to standard protocols.

NlM-like DNA fragments are obtained from all plant species tried. In many cases, only one NIM-like sequence was obtained. Table 3 and FIG. 2 detail the isolated NlH-like fragments. Table 3: NlH-like PCR fragments

[Table 3]

[0110] Based on these results, the degenerate primer PCR described above can amplify NlM-like fragments from a wide range of plant species. In particular, with the NIM 2B / NIM 2D primer combination,
All attempted plant species were successful using cDNA as a template. The use of ready-to-use PCR beads is particularly preferred for obtaining the product. Furthermore, if genomic DNA is sufficient, it is preferred to use cDNA as a template for all samples except Arabidopsis, tomato and sugar beet.

Example 7: Additional degenerate primers A new set of degenerate primers was used to determine whether tomatoes also contained similar NIM-like sequences that were not amplified using the degenerate primers listed in Table 1. To use
Designed based on the sequence alignment of four tobacco fragments (SEQ ID NO: 29, 31, 33 and 35) and tomato sequence (SEQ ID NO: 37). The primers designed from these fragments are described in Table 3. Genosys Biotechnologies, Inc. (The Woodlands, Te
xas). The degenerate sites are indicated in Table 3 by the notation of one or more bases at a single site in the oligonucleotide. “Orientation” indicates whether the primer is oriented in the 3 ′ end (downstream) direction or the 5 ′ end (upstream) direction of the cDNA.

Table 4: Additional degenerate primers

[Table 4]

[0113] Degenerate primer PCR was performed as described above using tomato cDNA to clone and sequence the latent product. Sequence analysis reveals a classification of the two NlM-like fragments. The first is identical to the sequence of the tomato shown in SEQ ID NO: 37, the second is unique in tomato, with 88% identity to the sequence of tobacco as shown in SEQ ID NOs: 31 and 33 there were. SEQ ID NO: 6 of this new tomato sequence
Shown as 1.

Example 8: Full-length NIM-like cDNA Upstream and downstream of the corresponding cDNA sequence from the NIM-like PCR fragment are SMART RACE cD
Preferably, it is obtained by RACE PCR using an NA amplification kit (Clontech, Palo Alto, CA). Preferably, at least three independent RACE products were sequenced for each 5'- or 3'-end to eliminate PCR errors. Sugar beet, sunflower B and tobacco, corresponding to the NIM-like PCR fragments shown in SEQ ID NOs: 39, 43 and 31
The resulting full-length cDNA sequences corresponding to the B NIM1 homologues were identified as SEQ ID NOs: 63, 65 and
Shown as 73.

The NIM-like Arabidopsis thaliana cDNAs corresponding to the NIM-like genomic sequences AtNMLc2 (SEQ ID NO: 15), AtNMLc4-1 (SEQ ID NO: 17) and AtNMLc4-2 (SEQ ID NO: 19) are: It is preferred to clone by RT-PCR. Arabidopsis thaliana
RNA is reverse transcribed using an oligo dT primer. Specific sense and antisense oligonucleotides designed to base the resulting first strand cDNA on the 5 'and 3' ends of the coding region of each genomic sequence (SEQ ID NO: 15, 17 and 19) Amplify by PCR using primers. PCR fragments of the expected size are cloned in the vector and sequenced to ensure that these cDNA clones match the NIM-like genomic sequence. C that matches the NIM-like genomic sequence AtNMLc2 (SEQ ID NO: 15)
The DNA sequence is shown as SEQ ID NO: 67; NIM-like genomic sequence AtNMLc4-2 (SEQ ID NO:
The full length cDNA sequence corresponding to (17) is shown as SEQ ID NO: 69; NIM-like genomic sequence A
The full length cDNA sequence corresponding to tNMLc4-2 (SEQ ID NO: 19) is shown as SEQ ID NO: 71.

Example 9: Northern blot According to Northern data, NIM-like clones of sugar beet (SEQ ID NOs: 39 and 6)
3) The expression of 3 to 7-fold increase after 100 μM or 300 μM BTH (benzo (1,2,3) thiadiazole-carbothioic acid S-methyl ester) treatment. Also, from the Northern data, the expression of the sunflower A NIM-like clone (SEQ ID NO: 41) is constitutive. Furthermore, according to Northern data, a NIM-like clone of sunflower B
The expression of (SEQ ID NOs: 43 and 65) increases 2-fold after 100 or 300 μM BTH treatment.

II. Expression of Gene Sequences of the Invention in Plants NIM1 homologs of the invention can be introduced into plant cells using conventional recombinant DNA techniques. In general, this involves inserting the coding sequence of the invention into an expression system in which the coding sequence is heterologous (ie, not normally present) using standard cloning methods known in the art. The vector contains sequences necessary for the transcription and translation of the sequence encoding the inserted protein. Many vector systems known in the art may be used, for example, plasmids, bacteriophages, viruses and other modified viruses. Suitable vectors include, but are not limited to, viral vectors, such as the lambda vector systems λgtl121, λgtl0 and Charon 4;
Plasmid vectors such as pB1211, pBR322, pACYC177, pACYC184, pAR,
pKK223-3, pUC8, pUC9, pUC18, pUC19, pLG339, pRK290, pKC37, pKC101, pCD
NAII; and other similar systems. Components of the expression system may be modified to increase expression. For example, truncated sequences, nucleotide substitutions or other modifications may be made. The expression system described herein can in fact be used to transform any cereal plant under suitable conditions. The transformed cells are
NIM1 homologs can be regenerated in whole plants so that NIM1 homologues increase SAR gene expression and enhance disease resistance in transgenic plants.

Example 10: Construction of a plant expression cassette A coding sequence intended for expression in transformed plants is first aggregated into an expression cassette after a suitable promoter that can be expressed in plants. The expression cassette can also include any other sequences necessary or selected for expression of the transgene. Such sequences are intended to target, without limitation, transcription terminators, exogenous sequences that enhance expression, such as introns, vital sequences, gene products to specific organs and cellular compartments. The sequence. These expression cassettes can be easily introduced into the following plant transformation vectors. The following is a description of the various components of a typical expression cassette.

[0119] 1. Promoter Selection of the promoter used in the expression cassette determines the spatial and temporal expression pattern of the transgene in the transformed plant. The selected promoter is
The transgene is expressed in specific cell types (eg, plant epidermal cells, mesophyll cells, root cortex cells) or in specific tissues or organs (eg, roots, leaves or flowers). The choice reflects the desired location for gene product accumulation. On the other hand, the selected promoter can drive gene expression under various inducing conditions. A promoter alters its strength, that is, its ability to promote transcription. Depending on the host cell system utilized, one of many suitable promoters may be used, including the native promoter of the gene. The following are non-limiting examples of promoters that can be used in the expression cassette.

A. Constitutive Expression, Ubiquitin Promoter: Ubiquitin is a gene product known to accumulate in many cell types, and its promoter can be found in transformed plants (eg, sunflower Binet et al., 1991; Maize-Christensen et al., 1989; and Arabidopsis-Norris et al., 19
93) isolated from several species for use in The corn ubiquitin promoter was developed in a transformed monocotyledonous plant system and its sequences and vectors constructed for the transformation of monocotyledonous plants are described in Patent Application Publication EP 0 342 926 (to Lubr
izol). Taylor et al. (1993) describe a vector containing the corn ubiquitin promoter and the first intron (pAHC25), and in a large number of monocot cell suspensions when introduced by microprojection / impact methods. Describes its high activity. The Arabidopsis ubiquitin promoter is particularly preferred for use with the NIM1 homologs of the present invention. The ubiquitin promoter is suitable for gene expression in both monocotyledonous and dicotyledonous transformed plants. Suitable vectors are pAHC25 or all derivatives of the transformation vectors described in this application, modified by the introduction of both a suitable ubiquitin promoter and / or intron sequence.

B. Constitutive expression, CaMV 35S promoter: The construction of plasmid pCGN1761 is described in patent application publication EP 0 392 225 (Example 23). pCGN1761 contains a "two" CaMV 35S promoter and a tml transcription terminator with a unique EcoRl site between the promoter and terminator, and has a pUC-type backbone. Eco present in addition to Notl and Xhol sites
A derivative of pCGN1761 containing an R1 site and having a modified polylinker is constructed. The derivative constructed pCGN1761 ENX. pCGN1761 ENX is expressed in transgene plants in 35
Useful for cloning cDNA sequences or coding sequences (including bacterial ORF sequences) into polylinkers for the purpose of their expression under the control of the S promoter. In order to transfer the complete 35S promoter coding sequence-tml terminator cassette of such a construct into a transformation vector as described below, the HindIII, SphI, Sall and Xbal sites 5 'to the promoter and the terminator. Against Xbal
, BamHI and BgII sites 3 '. In addition, the two 35S promoter fragments were replaced with HindIII, SphI, Sall, Xbal for replacement with another promoter.
Alternatively, 5 'excision with Pstl and a polylinker restriction site (EcoRI, Notl or Xho
It can be removed by 3 'excision in any of l). If desired, modifications around the cloning site may be made by introducing sequences that can enhance translation. It is particularly useful when multiple expression is required. For example, pCGN1761 ENX is a US Patent No. 5,6
It can be modified by optimization of the translation start site as described in Example 37 of 39,949.

C. Constitutive expression, actin promoter Several isoforms of actin are known to be expressed in most cell types,
Thus, the actin promoter is a good choice for a structural promoter. In particular, promoters from the rice Actl gene have been replicated and characterized (Mc
Elroy et al., 1990). The 1.3 kb fragment of the promoter was found to contain all the regulatory sequences required for expression in rice protoplasts. In addition, numerous expression vectors based on the Actl promoter have been specifically constructed for use in monocotyledonous plants (McElroy et al. (1991)). These are introduced with Actl-Intron 1, Adhl 5 'flanking sequence and Adhl-Intron 1 (from maize alcohol dehydrogenase) and sequences from the CaMV 35S promoter. Vectors showing very high expression are 35S and Actl intron or Act
l was a fusion of the 5 'flanking sequence and the Actl intron. Optimization of sequences surrounding the starting ATG (of the GUS reporter gene) also enhances expression. The promoter expression cassette described by McElroy et al. (1991) can be easily modified for gene expression, and is particularly suitable for use in monocot hosts. For example, the promoter-containing fragment may be removed from the McElroy construct and used to replace the two 35S promoters in pCGN1761 ENX and subsequently be used to insert specific gene sequences. That is, the constructed fusion gene can be transferred to an appropriate transformation vector. In another report, the rice Actl promoter with its first intron was found to show high expression in cultured wheat cells (Chibbar et al., 1993).
).

D. Inducible Expression, PR-1 Promoter The two 35S promoters in pCGN1761 ENX can be replaced with another promoter that produces high expression levels. By way of example, US Patent No. 5,
One of the chemically regulated ones described in 614.395 can be replaced with two 35S promoters. The choice of the promoter is preferably cut out from its source by a restriction enzyme, but it is also possible to carry out PCR amplification using a primer having an appropriate terminal restriction site. If PCR-amplification is performed, the promoter should be resequenced after cloning of the amplified promoter in the target vector to check for amplification errors. Chemical / pathogen tunable tobacco PR-
The 1a promoter was isolated from plasmid pCIB1004 (for construction, EP 0 332
104 Example 21) and the plasmid pCGN1761ENX (Uknes et al., 1992). pCIB1004 was cut with Ncol and the 3 ′ overhang of the resulting linear fragment was blunted by treatment with T4 DNA polymerase. Then, the fragment was digested with Hindlll, and the obtained fragment containing the PR-1a promoter was purified by gel and isolated from pCGN1761ENX from which two 35S promoters had been removed. This was accomplished by digestion with Xhol, blunting with T4 polymerase, followed by digestion with HindIII, and isolation of a large vector terminator containing the isolated pCIB1004 promoter fragment. This results in pCGN1761E with Pur-1a promoter and tml terminator and an intervening polylinker with EcoRl and Notl sites.
This gave the NX derivative. The selected coding sequence can be inserted into this vector, and the fusion product (ie, promoter-gene-terminator) can continuously transform the following. It can be transferred to all sequentially selected transformation vectors that include it. A variety of chemical modulators, including the benzothiadiazole, isonicotinic acid, and salicylic acid compounds described in US Pat. .

E. Inducible Expression, Ethanol-Inducible Promoters Promoters that can be induced by certain alcohols or ketones, such as ethanol, may be used to provide inducible expression of a coding sequence of the present invention.
Such a promoter is, for example, the alcA gene promoter from Aspergillus nidulans (Caddick et al., 1998). In A. nidulans, the alcA gene encodes alcohol dehydrogenase I, whose expression is regulated by the AlcR transcription factor in the presence of a chemical inducer. For the purposes of the present invention, plasmids
CAT coding sequence in palcA: CAT containing the alcA gene promoter sequence fused to a minimal 35S promoter (Caddick et al., 1998) is a code of the invention that forms an expression cassette with the coding sequence under the control of the alcA gene promoter. Replaced by an array. This is done in a manner known to those skilled in the art.

F. Inducible Expression, Glucocorticoid-Inducible Promoter Induction of NIM1 homologue expression of the present invention using a steroid hormone-based system is also contemplated. For example, using an induction system mediated by glucocorticoid (Aoyama an
d Chua, 1997), gene expression was induced by application of glucocorticoids, for example, synthetic glucocorticoids, preferably dexamethasone, at a concentration of preferably 0.1 mM to 1 mM, more preferably 10 mM to 100 mM. For the purposes of the present invention, the luciferase gene sequence is formed under the control of six copies of the GAL4 upstream activation sequence fused to a minimal 35S promoter to form an expression cassette with the gene sequence encoding the NIM1 homolog. For this purpose, it is replaced by a gene sequence encoding a NIM1 homolog. This is done in a manner well known to those skilled in the art. Trans-act factor is a hormone-binding domain of rat glucocorticoid receptor (Picard et al.,
Herpesvirus protein VP16 fused to 1988) (Triezenberg et al., 1988).
A GAL4 DNA binding domain fused to the transactivation domain of (Keegan e
t al., 1986). Expression of the fusion protein is controlled by any suitable promoter for expression in plants known in the art or described herein. This expression cassette is contained in plants containing the gene sequence encoding the NIM1 homolog fused to the 6xGAL4 / minimal promoter. That is, the tissue or organ specificity of the fusion protein is achieved and the inducible NIM 1 homologous tissue or organ specificity is obtained.

G. Root-specific expression Another pattern of gene expression is expression in roots. A suitable root promoter is
It is also described in Framond (1991), published patent application EP 0 452 269. This promoter is inserted into a suitable vector for insertion of the selection gene, such as pCGN1761 ENX.
And then the complete promoter-gene-terminator cassette is introduced into the transformation vector of interest.

H. Damage Inducible Promoters Damage inducible promoters are suitable for gene expression. Numerus such
Many such promoters are described below (e.g., Xu et
al., 1993); Logemann et al., 1989; Rohrmeier & Lehle, 1993; Firek et al.
, 1993; Warner et al., 1993), all of which are suitable for use in the present invention. Logemann et al. Described the 5 'upstream sequence of the monocotyledon potato wun1 gene. X
u et al. show that a damage-inducible promoter (pin2) from a monocot potato is active in rice of a monocot plant. In addition, Rohrmeier & Lehle describes the cloning of induced damage and maize Wipl cDNA, which can be used to isolate homologous promoters using standard techniques. Similarly, Firek et al. A
nd Warner et al. describes a damage-inducing gene from the monocotyledonous plant Asparagus officinalis that is expressed at the site of local injury and pathogen entry. Cloning techniques are well known to those skilled in the art, and these promoters are introduced into a suitable vector,
It can be used to express the genes at the site of plant damage by binding to the genes according to the invention.

I. Soft Tissue (Pith)-Preferred Expression Patent application WO 93/07278 describes the isolation of a maize trpA gene that is preferentially expressed in soft tissue cells. The gene sequence and promoter extending from the transcription start site to 1726 bases are presented. Using standard molecular biotechnology techniques, this promoter or a portion thereof replaces the 35S promoter and can be used to drive the expression of foreign genes in the parenchyma (medullary) -preferred manner. For example, pCGN1761 can be transduced. In fact, fragments containing the parenchyma (medullary) -preferred promoter can be transformed into all vectors and modified for use in transformed plants.

J. Leaf-specific expression maize gene encoding phosphoenol carboxylase (PEPC)
Are described by Hudspeth & Grula (1989). Using standard molecular biotechnology techniques, the promoter for this gene is used to drive expression of all genes in transformed plants in a leaf-specific manner.

K. Pollen-specific expression WO 93/07278 describes the isolation of a maize calcium-dependent protein kinase (CDPK) gene expressed in pollen cells. The gene sequence and promoter extend up to 1400 bases from the start of transcription. Using standard molecular biotechnology techniques, the promoter, or a portion thereof, can be replaced with a 35S promoter and used to drive expression of a NIM1 homolog of the invention in a pollen-specific manner.

2. Transcription Terminators A variety of transcription terminators are available for use in expression cassettes. These are involved in the translation arrest over the transgene and its correct polyadenylation. Suitable transcription terminators are known for their function in plants,
Includes MV 35S terminator, tml terminator, opaline synthesis terminator and pea rbcS E9 terminator. They can be used in both monocots and dicots. In addition, a negative transcription terminator for the gene may be used.

3. Sequences for increasing or regulating expression Many sequences have been found to increase gene expression from within the transcription unit, and these sequences increase their expression in transformed plants. Therefore, it can be used together with the gene of the present invention. Various intron sequences have been shown to increase expression, especially in monocot plant cells. For example, the intron of the corn Adhl gene, when introduced into corn cells, significantly increases the expression of the wild-type gene under its cognate promoter. Intron 1 increased expression and was found to be particularly effective in fusion constructs with the chloramphenicol acetyltransferase gene (Callis et al., 1987). In the same experimental system, corn bro
Introns from the nze 1 gene had a similar effect in increasing expression.
Intron sequences are commonly incorporated into plant transformation vectors, generally a non-translated leader.

Many non-translated leader sequences derived from viruses are known to increase expression, and are particularly effective in dicotyledonous cells. In particular, tobacco mosaic virus (Tabako Tobacco Mosaic Virus) (TMV, "W-sequence"), maize chlorotic mott virus (MCMV), alfalfa mosaic virus (Alfalfa Mosaic Virus) (AMV) It has been shown to be effective in increasing (eg, Gallie et al., 1987; Skuzeski et al., 1990).

4. Targeting Intracellular Gene Products It is known that a variety of mechanisms for targeting gene products exist in plants and sequences that control the functioning of these mechanisms. For example, targeting of gene products to chloroplasts is controlled by signal sequences found at the amino acid termini of various proteins that are cleaved during chloroplast incorporation and produce mature proteins (e.g., Comai et al., 1988). These signal sequences are
Fused with foreign gene products, may enable import of foreign products in chloroplasts (
van den Broeck, et al., 1985). DNA coding for appropriate signal sequence
Encodes RUBISCO protein, CAB protein, EPSP synthase, GS2 protein and many other proteins known to be localized in chloroplasts
It can be isolated from the 5 'end of cDNAs (see the section entitled "Expression With Chloroplast Targeting" in Example 37 of US Patent No. 5,639,949).

Other gene products localize to other organs such as mitochondria and peroxisomes (eg, Unger et al., 1989). The cDNAs encoding these products can be engineered to target foreign gene products to these organs. Examples of such sequences are nuclear-encoded ATPases and aspartate aminotransferase isoforms specific for mitochondria. Targeting of cellular proteins has been described by Rogers et al. (1985).

In addition, sequences that cause targeting of the gene product to other cellular compartments have been characterized. Amino-terminal sequences are involved in targeting extracellular secretory granules from ER, apoplast, and aleurone (Koehler & Ho, 1990). In addition, the amino-terminal sequence in association with the carboxy-terminal sequence is involved in vacuolar targeting of the genetic product (Shinshi et al.
al., 1990).

By fusion of the appropriate targeting sequence described above to the transgene sequence of interest, it is possible to direct the transgene product to any organ or cellular compartment. For chloroplast targeting, for example, a chloroplast signal sequence from the RUBISCO gene, CAB gene, EPSP synthase gene or GS2 gene is fused in frame to ATG at the amino acid terminus of the transgene. The signal sequence chosen should include the known cleavage site and should account for any amino acids behind the cleavage site necessary to cleave the constructed fusion. In some cases, this requirement can be met by adding a few amino acids between the cleavage site and the transgene ATG, or replacing any number of amino acids in the transgene sequence. Fusions constructed for the import of chloroplasts were obtained from Bartlett et al. (1
982) and chloroplast uptake using the technique described by Wasmann et al. (1986), the efficiency of chloroplast uptake by translation in vitro by the in vitro transcribed construct was tested. These construction techniques are known in the art and can be applied to mitochondria and peroxisomes as well.

The mechanism described above for cellular targeting is based on the translational regulation of the promoter with a pattern that differs from the expression pattern of the promoter from the targeting signal derivative. Can be used not only for binding to an exogenous promoter but also for binding to an exogenous promoter.

Example 11: Construction of Plant Transformation Vectors Many transformation vectors that can be used to transform plants are known to those of ordinary skill in plant genetic engineering and are suitable for the present invention. Genes can be used in conjunction with all such vectors. The choice of vector will depend on the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers are preferred. Selectable markers commonly used during transformation are the nptll gene (Messing & Vierra, 1982; Bevan et al., 1983), which confers resistance to kanamycin and related antibiotics, and the herbicide phosphinothricin. Bar gene (Wh
ite et al., 1990; Spencer et al., 1990), the hph gene (Blochinger & Diggelmann) and metatrexate (metha), which confer resistance to the antibiotic hygromycin.
trexate) (Bourouis et al., 1983) and the EPSPS gene (US Patent Application No. 4, conferring glyphosate resistance).
940,935 and 5,188,642).

1. Vectors Suitable for Agrobacterium Transformation Many vectors are utilized for transformation using Agrobacterium tumefaciens. Usually, they carry at least one T-DNA border sequence and contain pBINl9 (Bev
an, Nucl. Acids Res. (1984)) and pXYZ. Below,
The construction of two exemplary vectors suitable for Agrobacterium transformation is described.

A. PCIB200 and pCIB2001: The binary vectors pcIB200 and pCIB2001 were used to construct recombinant vectors for use in Agrobacterium and were constructed in the following manner. pTJS75ka
n is digested with Narl (degradation) of pTJS75, which cleaves the tetracycline resistance gene
Schmidhauser & Helinski, 1985) followed by insertion of an Accl fragment derived from pUC4K with NPTII (Messing & Vierra, 1982; Bevan et al., 1983; McBride et al.,
1990) Created. Xhol linkers have left and right T-DNA borders, plant selectivity nos / npt
ll binds to the EcoRV fragment of PCIB7 containing the chimeric gene and pUC polylinker (Rothst
ein et al., 1987), Xhol digested fragments were digested with Sall to generate pCIB200
Replicated in pTJS75kan (see EP 0 332 104, Example 19). pCIB200 contains the following unique polylinker restriction sites: EcoRI, Sstl, Kpnl, Bill, Xbal and Sa.
ll. pCIB2001 is further constructed by inserting restriction sites into the polylinker
It is a derivative of pCIB200. The unique restriction site in the polylinker of pCIB2001 is EcoRl
, Sstl, Kpnl, Bglll, Xbal, Sall, Mlul, Bcll, Avrll, Apal, Hpal and Stul
It is. In addition, pCIB2001, which contains these unique restriction sites, is useful for plant and bacterial kanamycin selectivity, left and right T-DNA borders for Agrobacterium-mediated transformation, activation between E. coli and other hosts. RK2-induced trfA
It has functions and OriT and OriV functions derived from RK2. The pCIB2001 polylinker is suitable for cloning plant expression cassettes containing their own regulatory signals.

B. Its pCIB10 and Hygromycin Selective Derivatives: Two vectors, pCIB10, provide kanamycin resistance and T-DN for selection in plants.
It encompasses the genes encoding the right and left border sequences of A and incorporates sequences from the broad host range plasmid pRK252 that can replicate in both E. coli and Agrobacterium. The construct was described by Rothstein et al. (1987). Grit
Various derivatives of pCIB10 incorporating the gene for hygromycin B phosphotransferase described in z et al., (1983) are constructed. These derivatives are either hygromycin alone (pCIB743) or hygromycin and kanamycin (pCIB715, pCIB715).
CIB717).

2. Vectors Suitable for Non-Agrobacterium Transformations Transformations without Agrobacterium tumefaciens can avoid the need for T-DNA sequences in the selected transformation vectors, thus eliminating vectors lacking these sequences. , And T-DNA sequences. Agrobacterium-independent transformation techniques include particle bombardment, protoplast uptake (eg, PEG and electroporation), and transformation by microinjection. The choice of the vector often depends on the preferred choice of the species to be transformed. The following describes the construction of an exemplary vector suitable for non-Agrobacterium transformation.

A. pCIB3064: pCIB3064 is a pUC-derived vector suitable for direct gene induction technology in combination with selection with the herbicide basta (or phosphinothricin). Plasmid pCIB246 contains the CaMV 35S promoter and CaMV 35S transcription terminator in an artificial fusion with the E. coli GUS gene, and is described in PCT published application W
O93 / 07278. The 35S promoter in this vector is 5 'of the start site.
Contains two ATG sequences. These sites are mutated using standard PCR techniques, such as removing ATG to create the restriction sites SspI and PvuII. The new restriction sites are 96 and 37 bp away from the unique SalI site and 101 and 42 bp away from the actual start site. The resulting derivative of pCIB246 is named pCIB3025. Next, the GUS gene is excised from pCIB3025 by digestion with SalI and SacI, the ends are blunted, and religated to produce plasmid pCIB3060. Plasmid pJIT82 was obtained from John Innes Centre, Norwich and was purchased from Streptomyces
The 400 bp SmaI fragment containing the bar gene from viridochromogenes is excised and inserted into the HpaI site of pCIB3060 (Thompson et al., 1987). This allows pCIB30
The pCIB3064 contains the bar gene under the control of the CaMV 35S promoter and terminator for herbicide selection, the gene for ampicillin resistance (E
and selection of unique sites SphI, PstI, HindIII, and
Includes polylinker with BamHI. This vector is suitable for cloning plant expression cassettes containing their own regulatory signals.

B. PSOG19 and pSOG35: pSOG35 is a transformation vector that utilizes the dihydrofolate reductase (DFR) of the E. coli gene as a selectable marker that confers resistance to methotrexate. PCR is used to amplify an intron 6 (-550 bp) from the 35S promoter (-800 bp) maize Adh1 gene and an 18 bp GUS untranslated leader sequence from pSOG10. A 250 bp fragment encoding the E. coli dihydrofolate reductase type II gene was also amplified by PCR, and these two PCR fragments were pUC19
PB1221 (containing vector backbone and nopaline synthase terminator)
Assemble with the SacI-PstI fragment from Clontech). Assembly of these fragments produced pSOG19, which was fused to intron 6 sequence.
Includes 5S promoter, GUS leader, DHFR gene and nopaline synthase terminator. Replacing the GUS leader of PSOG19 with a leader sequence from maize Chlorotic Mottle virus (MCMV) produces the vector pSOG35. PS
OG19 and pSOG35 carry the pUC gene for ampicillin resistance and have HindIII, SphI, PstI and EcoRI sites available for cloning foreign materials.

Example 12: Transformation Once the gene sequence of interest has been cloned into an expression system, it is introduced into plant cells. Methods for plant transformation and regeneration are well known in the art. For example, Ti plasmid vectors, and direct DNA uptake, liposomes, electroporation, microinjection, and microfiring have been used to carry foreign DNA. Furthermore, plant cells can be transformed using bacteria of the genus Agrobacterium. The following is a description of representative techniques for transforming both dicotyledonous and monocotyledonous plants.

[0147] 1. Transformation of Dicotyledons Plant transformation techniques for dicotyledons are well known in the art and include Agrobacterium-based techniques and techniques that do not require Agrobacterium. Non-Agrobacterium technology involves direct uptake of exogenous genetic material by protoplasts or cells. This can be achieved by uptake mediated by PEG or electroporation, delivery mediated by particle bombardment, or by microinjection. Examples of these techniques are described in Paszkowski et al., 1984; Potrykus et al., 1985; Reich et al., 198.
6; and Klein et al., 1987. In each case, the transformed cells are regenerated into whole plants using standard techniques known in the art.

Agrobacterium-mediated transformation is a preferred technique for the transformation of dicotyledonous plants due to its high transformation efficiency and widespread use for many different species. Agrobacterium transformation is typically performed by having a host Agrobacterium strain carry a binary vector (e.g., pCIB200 or pCIB2001) having the foreign DNA of interest either on the co-located Ti plasmid or on the chromosome. Including introducing into a suitable Agrobacterium strain that may rely on complementation of the vir gene (e.g., strain CIB5 for pCIB200 and pCIB2001).
42 (Uknes et al., 1993). Introduction of the recombinant binary vector into Agrobacterium is carried out using E. coli having the recombinant binary vector, a plasmid such as pRK2013, and a helper E that can transfer the recombinant binary vector to the target Agrobacterium strain. This is achieved by a method that combines three parents using E. coli strains. Alternatively, this recombinant binary vector can be introduced into Agrobacterium by DNA transformation (Hofgen and Willmitzer, 1988).
).

Transformation of a target plant species with a recombinant Agrobacterium typically involves co-culturing Agrobacterium with explants from the plant and follows protocols well known in the art. The transformed tissue is the binary plasmid T-
Regenerate on selective media with antibiotic or herbicide resistance markers present between the DNA boundaries. Another approach to transforming plant cells with certain genes involves forcing inert or biologically active particles into plant tissues and cells. This technique is disclosed in U.S. Patent Nos. 4,945,050, 5,360,006, and 5,100,792. Generally, the method involves driving the inert or bioactive particles into the cell under conditions effective to penetrate the outer surface of the cell and effect its uptake therein. When using inert particles, the vector can be introduced into cells by coating the particles with a vector containing the desired gene. Alternatively, the vector may surround the target cell, such that the traces of the particles carry the vector into the cell. Bioactive particles (eg, dried yeast cells, dried bacteria or bacteriophage each containing the DNA to be introduced) can also be driven into the plant cell tissue.

1. Transformation of monocotyledonous plants Transformation of most monocotyledonous plant species is also now routine. Preferred techniques include direct gene transfer into protoplasts using PEG or electroporation techniques, and particle bombardment into callus tissue. Transformation can be contemplated with a single DNA species or multiple DNA species (ie, co-transformation), both of which techniques are suitable for use in the present invention. Co-transformation may have the advantage of avoiding complete vector assembly and generating a transgenic plant with unlinked loci for the gene in question and the selectable marker, so that if it is desired If so, it becomes possible to remove the selectable marker in subsequent generations. However, a disadvantage of using co-transformation is that the frequency of separate DNA species being integrated into the genome is less than 100% (Schocher et al., 1986).

Patent applications EP0292435, EP0392225, and WO93 / 07278 disclose the preparation of callus and protoplasts from selected inbred maize, transformation of protoplasts using PEG or electroporation, and maize plants from transformed protoplasts Describes techniques for reproduction. Gordon-Kamm et al. (1990) and F
(1990) have published a technique for the transformation of corn lines derived from A188 using particle bombardment. Further, WO 93/07278 and Koziel et al. (1993) describe techniques for the transformation of selected inbred maize by particle bombardment. This technique works with 1.5-2.5mm cut from corn ears 14-15 days after pollination
It utilizes a long immature corn embryo and a PDS-1000He Biolistics instrument for collisions.

Transformation of rice can also be performed by direct gene transfer techniques utilizing protoplasts or particle bombardment. Transformations mediated by protoplasts have been described for the Japonica and Indica types (Zhang et al., 1988; S.
himamoto et al., 1989; Datta et al., 1990). Both types can be transformed using particle bombardment in a conventional manner (Christou et al., 1991). Further, WO 93/21335 describes a technique for rice transformation by electroporation.

Patent application EP 0 332 581 describes a technique for the production, transformation and regeneration of Pooideae protoplasts. These techniques allow for the transformation of Dactylis and wheat. In addition, transformation of wheat using particle bombardment of cells of type C long-term renewable callus was performed by Vasil et al. (1992), and by wheat bombardment using callus bombardment induced by immature and immature embryos. The conversion has been described by Vasil et al. (1993). However, a preferred technique for wheat transformation involves the transformation of wheat by particle bombardment of immature embryos, and involves either a high sucrose or high maltose step prior to gene transfer. Prior to collision, 3% sucrose (Murashiga and Skoog, 1962) and 3m for induction of somatic embryos
An arbitrary number of embryos (0.75-1 mm in length) are plated on an MS medium containing g / L of 2,4-D, and the embryos are advanced in the dark. On the day of the selected bombardment, the embryos are removed from the induction medium and placed on an osmotic modulator (ie, an induction medium containing the desired concentration, typically 15% sucrose or maltose). The embryos are allowed to protoplasm for 2-3 hours and then bombarded. Twenty embryos per target plate are typical, but not critical.
Plasmids containing the appropriate gene (eg, pCIB3064 or pSG35) are precipitated on micrometer-sized gold particles using standard methods. Embryos on each plate, ~ 1000ps
Shoot with a DuPont Biolistics ™ helium device using an explosion pressure of i, a standard 80 mesh screen. After the bombardment, the embryos are returned to the dark and allowed to recover for about 24 hours (still on the osmotic modulator). After 24 hours, the embryos are removed from the osmotic modulator and returned to the induction medium, where they are held for about one month before regeneration. Approximately one month later, explants of embryos with growing embryogenic calli were replaced with a suitable selection agent (10 mg / L basta for pCIB3064,
In the case of pSOG35, a regeneration medium (MS) further containing 2 mg / L methotrexate)
+ 1 mg / L NAA, 5 mg / L GA). After approximately one month, the grown shoots are transferred to a larger sterile container known as "GA7" containing half strength MS, 2% sucrose, and the same concentration of selective agent. Transformation of monocotyledonous plants using Agrobacterium has also been described.
See WO94 / 00977 and U.S. Patent No. 5,916,616.

III. Breeding and Seed Production Example 13: Breeding Plants obtained by transformation with the genes of the present invention may be any of a variety of plant species, including monocots and dicots. However, the plants used in the method according to the invention are preferably selected from the list of agroeconomically important target crops disclosed above. The expression of the gene according to the invention in combination with other properties important for production and quality can be incorporated into plant lines by breeding. Breeding approaches and techniques are known in the art. For example,
See Welsh JR (1981); Ed. Wood DR (1983); Mayo O. (1987); Singh, DP (1986); and Wricke and Weber (1986).

The genetic properties integrated in the transgenic seeds and plants described above are transmitted by sexual reproduction or plant growth and can thus be maintained and transmitted to progeny plants. In general, such maintenance and propagation utilize known agricultural methods that have been developed to meet a particular purpose, such as cultivation, sowing or harvesting. Specialized processes, such as hydroponics or greenhouse technology, can also be applied.
Growing crops are vulnerable to attack and damage caused by insects or infections, and to competition by weeds, which results in weeds, plant diseases, insects, nematodes, and other harmful conditions. Measures are taken to control yield and improve yield. These include mechanical means such as cultivation of soil or removal of weeds and infected plants,
And the application of pesticides such as herbicides, fungicides, gameticides, nematicides, growth regulators, ripening agents and pesticides.

The advantageous genetic properties of the transgenic plants and seeds according to the invention can also be used for breeding plants, which are resistant to pests, herbicides or stress, improve nutritional value, yield The aim is to develop plants with improved properties, such as increased plant growth or improved structure with less loss from lodging or destruction. The various breeding steps are characterized by distinct human intervention, such as the selection of lines to be crossed, the control of pollination of parent lines, or the selection of appropriate progeny plants. Different breeding measures are taken depending on the properties desired. Related techniques are well known in the art and include hybridization, inbreeding, backcrossing, multiline breeding, variant fusion, interspecies hybridization, aneuploid techniques, etc.
However, it is not limited to these. Hybridization techniques further include sterilizing the plants to obtain male or female sterile plants by mechanical, chemical, or biochemical means. Cross-pollinating a male sterile plant with pollen from a different line ensures that the genomes of the male sterile and female fertile plants equally acquire the properties of the parental line. Thus, the transgenic seeds and plants according to the present invention can increase the effectiveness of conventional methods, such as, for example, herbicide or pesticide treatment, or obviate the need for such methods due to their modified genetic properties, It can be used for breeding improved plant lines. Aside from this,
New crops with improved stress tolerance can be obtained, which are better than products that could not tolerate equivalent harmful growth conditions due to their optimized genetic "equipment" Produces a quality harvest product.

Example 14: Seed production In seed production, seed germination excellence and homogeneity are essential properties of products, while seed germination excellence and etc. of seeds harvested and sold by farmers. Quality is not important. Due to the difficulty of keeping the crops free from seeds of other crops and weeds, controlling the disease that arises from the seeds, and producing seeds that germinate well, it is rather large and well defined. Seed production practices are being developed by seed producers, who have gained experience in the field of growing, improving and buying and selling pure seeds. Therefore, it is customary for farmers to purchase guaranteed seeds that meet clear quality standards without using seeds harvested from their crops. Propagation materials used as seed are conventionally treated with a protective coating comprising herbicides, insecticides, fungicides, fungicides, nematocides, slug repellents, or mixtures thereof. Conventionally used protective coatings are captan, carboxin, thyram (TMTDTM)
, Metalaccil (Apron ™), and pirimiphos-methyl (Actellic ™). If desired, these compounds are formulated with further carriers, surfactants or application aids conventionally used in the field of formulation to provide protection against damage caused by bacteria, fungi or animal pests.
The protective coating can be applied by impregnating the propagation material with the liquid formulation, or by coating with a combined wet or dry formulation. Other applications, such as treatment on shoots or fruits, are also possible.

It is a further aspect of the present invention to provide a new agricultural method, such as the above-exemplified method featuring the use of a transgenic plant, transgenic plant material, or transgenic seed according to the present invention. is there.

Seeds can be provided in bags, containers or containers containing suitable packaging material, which bags or containers can be closed to hold the seeds. Bags, containers or containers can be designed for either short or long term storage of seeds, or both. Examples of suitable packaging materials include paper, such as kraft paper, rigid or flexible plastic or other polymeric materials, glass or metal. Desirably, the bag, container, or container includes multiple layers of the same or different types of packaging material. In some embodiments, a bag,
Containers or containers are provided to exclude or limit water and moisture from contacting the seeds. In one example, the bag, container, or container is hermetically sealed, eg, heat sealed, to prevent water or moisture from entering. In another aspect, the water-absorbing material is located between or near the layers of packaging material. In yet another aspect,
The bag, container or container, or the packaging material comprising it, is treated to limit, control or prevent disease, contamination or other harmful effects on the storage or transport of the seed. One example of such a treatment is sterilization, for example by chemical means or irradiation. Claims 1. A commercial bag comprising seeds of a transgenic plant comprising a gene according to the invention, together with a suitable carrier, wherein said gene is expressed at a higher level in said transformed plant than in a wild-type plant. Bags that together with descriptive labels for their use to confer broad spectrum disease resistance to
Included in the present invention.

IV. Assessment of Disease Resistance Assessment of disease resistance is performed by methods known in the art. Uknes et al. (1993); Gorla
See Ch. et al. (1996); Alexander et al. (1993). For example, some representative assays for disease resistance are described below.

Example 15: Phytophthora parasitica (tobacco plague) resistance assay A test for resistance to Phytophthora parasitica, the causative agent of tobacco plague, is performed on 6-week-old plants grown as described by Alexander et al. (1993). . Plants are irrigated, drained well, and then inoculated by applying 10 mL of the sporangia suspension (300 sporangia / mL) to the soil. Plants inoculated are daytime temperature 23-25 ° C, nighttime temperature 20-22
Keep in greenhouse maintained at ° C. The wilting indicators used in the test are as follows: 0 =
No symptoms; 1 = No symptoms; 1 = Some signs of wilt with reduced tone; 2 = Symptoms of wilt, but without rot or growth inhibition; 3 = Occurring wilt with growth inhibition Symptoms, but apparently no stem rot; 4 = severe wilt, with visible stem rot and some damage to root system; 5 = same as 4, except that plant is dead or near death and severe rhizome With significant impairment. All assays are scored mechanically on plants arranged in a random design.

[0162] Example 16:. Pseudomonas syringae resistance test Pseudomonas syringae pv the tabaci strain # 551, at a concentration in the water 10 ⁶ or 3x10 ⁶ / mL, injected into two lower leaves of several 6-7 week old plants . Six individual plants are evaluated at each time point. Plants infected with Pseudomonas tabaci were scored on a 5-point disease severity scale (5 =
100% dead tissue, 0 = no symptoms). T-test (
LSD) and order grouping according to average disease grade values. The numbers that have the same letter on the date of the assessment are not statistically significantly different.

Example 17: Cercospora nicotianae resistance assay A spore suspension of Cercospora nicotianae (ATCC # 18366) (100,000-150,000 spores per mL) is sprayed on the leaf surface and immediately allowed to run off. The plant is maintained at 100% humidity for 5 days. The plant is then sprayed with water 5-10 times / day. Six individual plants are evaluated at each time point. Cercospora nicotianae is graded based on% area of leaf showing disease symptoms. A T-test (LSD) is performed for each day assessment and indicates grouping according to the average disease grade value. The numbers that have the same letter on the date of the assessment are not statistically significantly different.

Example 18: Peronospora parasitica resistance assay A resistance assay for Peronospora parasitica is performed on plants as described in Uknes et al. (1993). By spraying a conidia suspension (approximately 5x10 ⁴ spores per milliliter) plants are inoculated with compatible isolate of P.Parasitica. The inoculated plants are incubated in a growth room at 17 ° C. under humid conditions, with a cycle of 14 hours day / 10 hours night. Plants are examined for the presence of conidiophores 3-14 days, preferably 7-12 days after inoculation. In addition, some of the treated plants are randomly selected and stained with lactophenol-trypan blue for microscopic observation (Keogh et al., 1980). The embodiments disclosed above are exemplary. The disclosure of the invention will provide those skilled in the art with access to numerous variations of the invention. All such obvious and foreseeable variations are intended to be covered by the appended claims.

BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTINGS SEQ ID NO: 1-full length cDNA sequence of NIM1 homologue from Nicotiana tabacum. SEQ ID NO: 2-Protein sequence of NIM1 homolog of Nicotiana tabacum encoded by SEQ ID NO: 1. SEQ ID NO: 3-Full length cDNA sequence of NIM1 homologue from Lycopersicon esculentum. SEQ ID NO: 4-NIM1 of Lycopersicon esculentum encoded by SEQ ID NO: 3
Homologous protein sequence. SEQ ID NO: 5-Partial cDNA sequence of NIM1 homologue from Brassica napus. SEQ ID NO: 6--Partial protein sequence of NIM1 homolog of Brassica napus encoding SEQ ID NO: 5. SEQ ID NO: 7-Full length cDNA of NIM1 homologue (AtNMLc5) from Arabidopsis thaliana
Array. SEQ ID NO: 8-full length protein sequence of Arabidopsis thaliana NIMl homolog AtNMLc5 encoded by SEQ ID NO: 7. SEQ ID NO: 9—14-oligonucleotide primer used in Examples 1-4. SEQ ID NO: 15—Genomic DNA sequence of NIM1 homologue (AtNMLc2) from Arabidopsis thaliana. SEQ ID NO: 16-NIM1 of Arabidopsis thaliana encoded by SEQ ID NO: 15
Protein sequence of the homolog AtNMLc2. SEQ ID NO: 17--Genomic DN of NIM1 homologue (AtNMLc4-1) from Arabidopsis thaliana
A array. SEQ ID NO: 18-Protein sequence of Arabidopsis thaliana NIM1 homolog AtNMLc4-1 encoded by SEQ ID NO: 17. SEQ ID NO: 19-Genomic DN of NIM1 homologue (AtNMLc4-2) from Arabidopsis thaliana
A array. SEQ ID NO: 20-NIM1 of Arabidopsis thaliana encoded by SEQ ID NO: 19
Protein sequence of homologue AtNMLc4-2. SEQ ID NO: 21-PCR primer-NIM 1A. SEQ ID NO: 22-PCR primer-NIM 1B. SEQ ID NO: 23-PCR primer-NIM 1C. SEQ ID NO: 24-PCR primer-NIM 1D. SEQ ID NO: 25-PCR primer-NIM 2A. SEQ ID NO: 26-PCR primer-NIM 2B. SEQ ID NO: 27-PCR primer-NIM 2C. SEQ ID NO: 28-PCR primer-NIM 2D. SEQ ID NO: 29--A 659 base NI amplified from Nicotiana tabacum (tobacco A) with 36 sequence matches and 67% sequence match with Arabidopsis thaliana NIM1 gene sequence
M-like DNA fragment. SEQ ID NO: 30-Protein sequence encoded by SEQ ID NO: 29. SEQ ID NO: 31—498 base NIM-like DNA fragment amplified from Nicotiana tabacum (tobacco B) in which the two sequences match and 62% match the Arabidopsis thaliana NIM1 gene sequence. SEQ ID NO: 32-Protein sequence encoded by SEQ ID NO: 31. SEQ ID NO: 33—NIM-like DNA fragment of 498 bases amplified from Nicotiana tabacum (tobacco C) in which three sequences match the Arabidopsis thaliana NIM1 gene sequence and 63% in sequence. SEQ ID NO: 34-Protein sequence encoded by SEQ ID NO: 33. SEQ ID NO: 35--Ni with a 59% sequence identity to the Arabidopsis thaliana NIM1 gene sequence
NIM-like DNA fragment of 399 bases amplified from cotiana tabacum (tobacco D). SEQ ID NO: 36-Protein sequence encoded by SEQ ID NO: 35. SEQ ID NO: 37-35—498 base NIM-like DNA fragment amplified from Lycopersicon esculentum (tomato A) having 67% sequence identity with Arabidopsis thaliana NIM1 gene sequence. SEQ ID NO: 38-Protein sequence encoded by SEQ ID NO: 37. SEQ ID NO: 39-24 A 498 base NIM-like DNA fragment amplified from Beta vulgaris (sugar beet) showing consensus on sequence and 66% sequence identity with the Arabidopsis thaliana NIM1 gene sequence. SEQ ID NO: 40-Protein sequence encoded by SEQ ID NO: 39. SEQ ID NO: 498 amplified from Helianthus annuus (Sunflower A) showing consensus of 9 sequences with the Arabidopsis thaliana NIM1 gene sequence and 61% sequence identity
NIM-like DNA fragment of base. SEQ ID NO: 42-Protein sequence encoded by SEQ ID NO: 41. SEQ ID NO: 43--Amplified from Helianthus annuus (Sunflower B) showing consensus of 10 sequences with the Arabidopsis thaliana NIM1 gene sequence and 59% sequence identity
NIM-like DNA fragment of 498 bases. SEQ ID NO: 44-Protein sequence encoded by SEQ ID NO: 43. SEQ ID NO: 45--Amplified from Solanum tuberosum (Potato A) 65, showing a 15 sequence consensus and 68% sequence identity with the NIM1 gene sequence of Arabidopsis thaliana
NIM-like DNA fragment of 3 bases. SEQ ID NO: 46-Protein sequence encoded by SEQ ID NO: 45. SEQ ID NO: 47--Amplified from Solanum tuberosum (Potato B) showing a consensus of 3 sequences and 61% sequence identity with the NIM1 gene sequence of Arabidopsis thaliana
NIM-like DNA fragment of base. SEQ ID NO: 48-Protein sequence encoded by SEQ ID NO: 47. SEQ ID NO: 49-Amplified from Solanum tuberosum (Potato C), showing consensus of 2 sequences and 62% sequence identity with NIM1 gene sequence of Arabidopsis thaliana
NIM-like DNA fragment of base. SEQ ID NO: 50-Protein sequence encoded by SEQ ID NO: 49. SEQ ID NO: 51--Amplified from Brassica napus (canola A) showing a consensus of 5 sequences and 59% sequence identity with the NIM1 gene sequence of Arabidopsis thaliana
One base NIM-like DNA fragment. SEQ ID NO: 52-protein sequence encoded by SEQ ID NO: 51. SEQ ID NO: 53--Amplified from Brassica napus (canola A) showing a consensus of 5 sequences and a sequence identity of 58% with the NIM1 gene sequence of Arabidopsis thaliana
One base NIM-like DNA fragment. SEQ ID NO: 54-Protein sequence encoded by SEQ ID NO: 53. SEQ ID NO: 55—498-base NIM-like DNA fragment amplified from Brassica napus (canola C) showing 56% sequence identity with the NIM1 gene sequence of Arabidopsis thaliana. SEQ ID NO: 56-Protein sequence encoded by SEQ ID NO: 55. SEQ ID NO: 57—NIM-like DNA fragment of 498 bases amplified from Brassica napus (canola D) showing 73% sequence identity with NIM1 gene sequence of Arabidopsis thaliana. SEQ ID NO: 58-Protein sequence encoded by SEQ ID NO: 57. SEQ ID NO: 59-PCR primer-NIM 3A. SEQ ID NO: 60-PCR primer-NIM 3B. SEQ ID NO: 61--NIM1 gene sequence of Arabidopsis thaliana, 148 bases NIM-like DNA fragment amplified from Lycopersicon esculentum (tomato B), showing a consensus of 3 sequences and 72% sequence identity. SEQ ID NO: 62-Protein sequence encoded by SEQ ID NO: 61. SEQ ID NO: 63-derived from Beta vulgaris (sugar beet) corresponding to the PCR fragment of SEQ ID NO: 39
Full length cDNA sequence of NIM1 homolog. SEQ ID NO: 64-Protein sequence of sugar beet NIM1 homologue encoded by SEQ ID NO: 62. SEQ ID NO: 65-Helianthus annuus corresponding to the PCR fragment of SEQ ID NO: 43 (Sunflower B)
Full length cDNA sequence of the NIM1 homologue from E. coli. SEQ ID NO: 66-Protein sequence of Helianthus annuus NIM1 homologue encoded by SEQ ID NO: 65. SEQ ID NO: 67-NIM-like genomic sequence of Arabidopsis thaliana AtNMLc2 (SEQ ID NO:
CDNA sequence corresponding to 15). SEQ ID NO: 68-Protein sequence encoded by SEQ ID NO: 67. SEQ ID NO: 69-cDNA sequence corresponding to NIM-like genomic sequence of Arabidopsis thaliana AtNMLc4-1 (SEQ ID NO: 17). SEQ ID NO: 70-Protein sequence encoded by SEQ ID NO: 69. SEQ ID NO: 71-cDNA sequence corresponding to NIM-like genomic sequence of Arabidopsis thaliana AtNMLc4-1 (SEQ ID NO: 19). SEQ ID NO: 72-Protein sequence encoded by SEQ ID NO: 71. SEQ ID NO: 73-Full length cDNA sequence of the NIM1 homologue from Nicotiana tabacum (tobacco B) corresponding to the PCR fragment of SEQ ID NO: 31. SEQ ID NO: 74-Protein sequence of NIM1 homolog of Nicotiana tabacum encoded by SEQ ID NO: 73.

Deposits The following materials are available under the authority of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Patent Procedure under the Agricultural Research Service, Patent Culture Collection (
NRRL), deposited at 1815 North University Street, Peoria, Illinois 61604, USA. All restrictions on the use of the deposit cannot be removed for patent grant.

Clone Accession Number Deposit Date pNOV1203 NRRL B-30049 August 17, 1998 pNOV1204 NRRL B-3050 August 17, 1998 pNOV1206 NRRL B-30051 August 17, 1998 AtNMLc5 NRRL B-30139 1999 May 25

References The references cited herein represent the state of the art. Each of the following documents is hereby incorporated by reference. USNo. 4,940,935 USNo.4,945,050 USNo.5,036,006 USNo.5,100,792 USNo.5,188,642 USNo.5,523,311 USNo.5,591,616 USNo.5,614,395 USNo.5,639,949 USNo. EP 0 452 269 International PCT Application WO 93/07278 International PCT Application WO 93/21335 International PCT Application WO 94/00977 International PCT Application WO 94/13822 International PCT Application WO 94/16077 International PCT Application WO 97/49822 International PCT Application WO 98/06748 International PCT Application WO 98/26082 International PCT Application WO 98/29537 Alexander et al., Proc. Natl. Acad. Sci. USA 90: 7327-7331 (1993) Aoyama and Chua, The Plant Journal 11: 605- 612 (1997) Ausubel, FM et al., Current Protocols in Molecular Biology, pub. By G
reene Publishing Assoc. and Wiley-Interscience (1987) Bartlett et al., In: Edelmann et al. (Eds.) Methods in Chloroplast Molecu
lar Biology, Elsevier pp 1081-1091 (1982) Bevan et al., Nature 304: 184-187 (1983) Bevan, Nucl. Acids Res. (1984) Bi et al., Plant J. 8: 235-245 (1995 Binet et al. Plant Science 79: 87-94 (1991) Blochinger & Diggelmann, Mol Cell. Biol. 4: 2929-2931 Bourouis et al., EMBO J. 2 (7): 1099-1104 (1983) Boutry et. al., Nature 328: 340-342 (1987) Caddick et aL, Nat.Biotechnol 16: 177-180 (1998) Callis et al., Genes Develop. 1: 1183-1200 (1987) Cameron et al., Plant J 5: 715-725 (1994) Cao et al., Plant Cell 6, 1583-1592 (1994) Cao et al., Cell 88: 57-63 (1997) Casas et al., Proc. Natl. Acad. Sci. USA 90: 11212-11216 (1993) Century et al., Proc. Natl. Acad. Sci. USA 92: 6597-6601 (1995) Chibbar et al., Plant Cell Rep. 12: 506-509 (1993) Chrispeels Plant Rev. Plant Physiol.Plant Mol bol 42: 21-53 (1991) Christou et al., Plant Physiol. 87: 671-674 (1988) Christou et al., Biotechnology 9: 957-962 (1991) Christensen et al., Plant Molec. Biol. 12: 619-632 (1989) Comai. et al., J. Biol. Chem. 263: 15104-15109 (1988) Crossway et al., BioTechniques 4: 320-334 (1986) Datta et al., Biotechnology 8: 736-740 (1990) de Framond, FEBS. 290: 103-106 (1991) Delaney et al., Science 266: 1247-1250 (1994) Delaney et al., Proc.Natl.Acad.Sci. USA 92: 6602-6606 (1995) Dempsey and Klessig, Bulletin de L'Institut Pasteur 93: 167-186 (1995) Dietrich et al., Cell 77: 565-577 (1994) Firek et al., Plant Molec.Biol. 22: 129-142 (1993) Fromm et al., Biotechnology 8: 833-839 (1990) Gaffney et al., Science 261: 754-756 (1993) Galle et al., Nucl. Acids Res. 15: 8693-8711 (1987) Glazebrook et al., Genetics 143: 973- 982 (1996) Gordon-Kamm et al., Plant Cell 2: 603-618 (1990) Gordon-Kamm et al, in "Transgenic Plants", vol. 2., pp. 21-33, pub.b
y Academic Press (1993) Goerlach et al., Plant Cell 8: 629-643 (1996) Greenberg et al, Cell 77: 551-563 (1994) Gritz et al., Gene 25: 179-188 (1983) Hayashimoto et al., Plant Physiol. 93: 857-863 (1990) Hill et al., Euphytica 85: 119-123 (1995) Hinchee et al., Biotechnology 6: 915-921 (1988) Hoefgen & Willmitzer, Nucl. Acids Res. 16: 9877 (1988) Hudspeth & Grula, Plant Molec.Biol 12: 579-589 (1989) Hunt and Ryals, Crit. Rev. in Plant Sci. 15: 583-606 (1996) Innis et al., PCR Protocols , a Guide to Methods and Applications eds.,
Academic Press (1990) Ishida et al., Nature Biotechnology 14: 745-750 (1996) Jahne et al., Theor.Appel.Genet. 89: 525-533 (1994) Keegan et al., Science 231: 699-704. (1986) Keogh et al., Trans.Br. Mycol.Soc. 74: 329-333 (1980) Klein et al., Nature 327: 70-73 (1987) Klein et al., Proc. Natl. Acad. Sci. USA 85: 4305-4309 (1988) Klein et al., Bio / Technology 6: 559-563 (1988) Klein et al., Plant Physiol. 91: 440-444 (1988) Koziel et al., Biotechnology 11: 194-200 (1993) Koziel et al., Annals of the New York Academy of Sciences 792: 164-171 (
1996) Lawton et al., "The molecular biology of systemic aquired resistance" in
Mechanisms of Defense Responses in Plants, B. Fritig and M. Legrand, eds
(Dordrecht, The Netherlands: Kluwer Academic Publishers), pp. 422-432
(1993) Lawton et al., Plant J. 10: 71-82 (1996) Logemann et al., Plant Cell 1: 151-158 (1989) Maher et al., Proc. Natl. Acad. Sci. USA 91: 7802-7806 (1994) Mauch-Mani and Slusarenko, Mol.Plant-Microbe Interact. 7: 378-383 (1
994) Mauch-Mani and Slusarenko, Plant Cell 8: 203-212 (1996) Mayo O., The Theory of Plant Breeding, Second Edition, Clarendon Press,
Oxford (1987) Mazur et al., Plant Physiol. 85: 1110 (1987) McBride et al., Plant Molecular Biology 14: 266-276 (1990) McCabe et al., Biotechnology 6: 923-926 (1988) McElroy et. al., Plant Cell 2: 163-171 (1990) McElroy et al., Mol.Gen. Genet. 231: 150-160 (1991) Messing & Vierra, Gene 19: 259-268 (1982) Murashiga & Skoog, Physiologia Plantarum 15: 473-497 (1962) Nehra et aL, The Plant Journal 5: 285-297 (1994) Neuhaus et al., Proc.Natl.Acad.Sci. USA 88: 10362-10366 (1991) Norris et al. , Plant Mol bol 21: 895-906 (1993) Pallas et al., Plant J. 10: 281-293 (1996) Parker et al., Plant Cell 8: 2033-2046 (1996) Paszkowski et al., EMBO J 3: 2717-2722 (1984) Payne et al., Plant Mol Biol. 11: 89-94 (1988) Picard et al., Cell 54: 1073-1080 (1988) Potrykus et al., Mol. Gen. Genet. 199: 169-177 (1985) Reich et al., Biotechnology 4: 1001-1004 (1986) Riggs et al., Proc.Natl.Acad.Sci. USA 83: 5602-5606 (1986) Rogers et al., Proc. Natl. Acad. Sci. USA 82: 6512-6516 (1985) Ro hrmeier & Lehle, Plant Molec.Biol. 22: 783-792 (1993) Rothstein et al., Gene 53: 153-161 (1987) Ryals et al., Plant Cell 8: 189-1819 (1996) Ryals et al. , Plant Cell 9: 425-439 (1997) Sambrook et al., Molecular Cloning, eds., Cold Spring Harbor Laboratory.
Press (1989) Sanford et al., Particulate Science and Technology 5: 27-37 (1987) Schmidhauser & Helinski, J. Bacteriol. 164: 446-455 (1985) Schocher et al., Biotechnology 4: 1093-1096 (1986) Shimamoto et al., Nature 338: 274-277 (1989) Shinshi et al., Plant Molec.Biol. 14: 357-368 (1990) Shulaev et al., Plant Cell 7: 1691-1701 (1995) Silhavy, et al., Experiments with Gene Fusions, eds., Cold Spring Harbo
r Laboratory Press (1984) Singh, DP, Breeding for Resistance to Diseases and Insect Pests, Spri
nger-Verlag, NY (1986) Skuzeski et al., Plant Molec.Biol. 15: 65-79 (1990) Somers et al., Bio / Technology 10: 1589-1594 (1992) Spencer et al., Theor.Appl Genet. 79: 625-631 (1990) Stanford et al., Mol. Gen. Genet. 215: 200-208 (1989) Svab et al., Proc. Natl. Acad. Sci. USA 87: 8526-8530 ( 1990) Taylor et al., Plant Cell Rep. 12: 491-495 (1993) Thompson et al. EMBO J. 6: 2519-2523 (1987) Torbert et al., Plant Cell Reports 14: 635-640 (1995) Triezenberg et al., Genes Devel. 2: 718-729 (1988) Uknes et al., Plant Cell 4: 645-656 (1992) Uknes et al. Plant Cell 5: 159-169 (1993) Uknes et al., Molecular Plant Microbe Interactions 6: 680-685 (1993) Uknes et al., Mol.Plant-Microbe Interact. 6: 692-698 (1993) Umbeck et al., Bio / Technology 5: 263-266 (1987) Unger et 13: 411-418 (1989) van den Broeck, et al., Nature 313: 358-363 (1985) Vasil et al., Biotechnology 10: 667-674 (1992) Vasil et al. ., Biotechnology 11: 1553-1558 (1993) Vernooij et al., Plan t Cell 6: 959-965 (1994) Vernooij et al., Mol.Plant-Microbe Interact. 8: 228-234 (1995) Von Heijne et al., Plant Mol Biol.Rep. 9: 104-126 (1991) Vorst et al., Gene 65:59 (1988) Wan et al., Plant Physiol. 104: 37-48 (1994) Ward et al., Plant Cell 3: 1085-1094 (1991) Warner et al., Plant J 3: 191-201 (1993) Wasmann et al., Mol.Gen. Genet. 205: 446-453 (1986) Weeks et al., Plant Physiol. 102: 1077-1084 (1993) Weissinger et al., Annual. Rev. Genet. 22: 421-477 (1988) Welsh JR, Fundamentals of Plant Genetics and Breeding, John Wiley & S
ons, NY (1981) Weymann et al., Plant Cell 7: 2013-2022 (1995) White et al., Nucl. Acids Res. 18: 1062 (1990) Wood DR (Ed.) Crop Breeding, American Society of Agronomy Maison, Wis
consin (1983) Wricke and Weber, Quantitative Genetics and Selection Plant Breeding,
Walter de Gruyter and Co., Berlin (1986) Xu et al., Plant Molec. Biol. 22: 573-588 (1993) Zhang et al., Plant Cell Rep. 7: 379-384 (1988)

[0169]

[Table 5]

[0170]

[Table 6]

[0171]

[Table 7]

[0172]

[Table 8]

[0173]

[Table 9]

[0174]

[Table 10]

[0175]

[Table 11]

[0176]

[Table 12]

[Sequence list]

──────────────────────────────────────────────────続 き Continuation of front page (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, TZ, 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, DM, 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, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72 Inventor Laura Jean Wayslo United States 27603 West South Street, Raleigh, North Carolina, No. 914 (72) Inventor Michael G. Willits United States 27502 Apex, North Carolina, Winter Hill Drive No. 804 (72) Inventor Person Tesfei Mengiste 27713 Penris Drive, Durham, North Carolina 5206 F-term (reference) 2B030 AA02 AB03 AD05 CA06 CA17 CA19 C D03 CD06 CD09 4B024 AA08 BA80 CA04 DA01 DA05 EA04 FA02 GA11 4B065 AA11X AA89X AA89Y AB01 AC20 CA53

Claims (23)

[Claims] (1) SEQ ID NO: 2, 4, 6, 8, 16, 18, 20, 30, 32, 34, 36, 38, 40, 42, 44,
Nucleotide sequences encoding 46, 48, 50, 52, 54, 56, 58, 62, 64, 66, 68, 70, 72 or 74; (b) SEQ ID NOs: 1, 3, 5, 7, 15, 17, 17 , 19, 29, 31, 33, 35, 37, 39, 41, 43,
45, 47, 49, 51, 53, 55, 57, 61, 63, 65, 67, 69, 71 or 73; (c) SEQ ID NOs: 1, 3, 5, 7, 15, 17, 19, 29, 31, 33, 35, 37, 39, 41, 43,
At least 2 of 45, 47, 49, 51, 53, 55, 57, 61, 63, 65, 67, 69, 71 or 73
A nucleotide sequence comprising a portion of at least 20 consecutive base pairs that are identical in sequence to 0 consecutive base pairs; (d) SEQ ID NOs: 9 and 10, SEQ ID NOs: 21 and 24, SEQ ID NO: 22 and 24,
Lycopersicon using the polymerase chain reaction with a set of primers set forth as SEQ ID NOs: 25 and 28, SEQ ID NOs: 26 and 28, or SEQ ID NOs: 59 and 60.
nucleotide sequences that can be amplified from an esculentum DNA library; (e) nucleotides that can be amplified from a Beta vulgaris DNA library using the polymerase chain reaction with a set of primers set forth as SEQ ID NOs: 22 and 24 or SEQ ID NOs: 26 and 28. (F) a nucleotide sequence that can be amplified from a Helianthus annuus DNA library using the polymerase chain reaction with a set of primers described as SEQ ID NOs: 26 and 28; (g) SEQ ID NOs: 21 and 24, SEQ ID NO: 21 and 23, SEQ ID NOs: 22 and 24,
Nucleotide sequences that can be amplified from a Solanum tuberosum DNA library using the polymerase chain reaction with a set of primers set forth as SEQ ID NOs: 25 and 28 or SEQ ID NOs: 26 and 28; (h) SEQ ID NOs: 9 and 10 or the sequences Nucleotide sequences that can be amplified from a Brassica napus DNA library using the polymerase chain reaction with a set of primers described as Nos. 26 and 28; (i) SEQ ID NOs: 13 and 14, SEQ ID NOs: 21 and 24 or SEQ ID NO: : 22 and
Arabid using the polymerase chain reaction with a set of primers described as 24
a nucleotide sequence that can be amplified from an opsis thaliana DNA library; (j) SEQ ID NOS: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 21 and 24,
Nicotiana ta using the polymerase chain reaction with a set of primers set forth as SEQ ID NOs: 22 and 24, SEQ ID NOs: 25 and 28 or SEQ ID NOs: 26 and 28
a nucleotide sequence that can be amplified from a bacum DNA library; or (k) the first 20 nucleotides and SEQ ID NOs: 1, 3, 5, 7, 15, 17, 19, 29
, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 61, 63, 65, 67
, A nucleotide sequence that can be amplified from a plant DNA library using the polymerase chain reaction with a set of primers comprising the reverse complement of at least 20 nucleotides of the coding sequence (CDS) of 69, 71 or 73; An isolated nucleic acid molecule. 2. SEQ ID NO: 2, 4, 6, 8, 16, 18, 20, 30, 32, 34, 36, 38,
40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 62, 64, 66, 68, 70, 72 or 74
2. The isolated nucleic acid molecule of claim 1, which comprises a nucleotide sequence that encodes: 3. SEQ ID NO: 1, 3, 5, 7, 15, 17, 19, 29, 31, 33, 35, 37
, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 61, 63, 65, 67, 69, 71 or 7
3. The isolated nucleic acid molecule of claim 1, comprising 3. 4. SEQ ID NO: 1, 3, 5, 7, 15, 17, 19, 29, 31, 33, 35, 37
, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 61, 63, 65, 67, 69, 71 or 73
2. A nucleotide sequence comprising a portion of at least 20 consecutive base pairs and a portion of at least 20 consecutive base pairs that are identical in sequence.
4. The isolated nucleic acid molecule of claim 1. 5. SEQ ID NO: 9 and 10, SEQ ID NO: 21 and 24, SEQ ID NO: 22 and 24, SEQ ID NO: 25 and 28, SEQ ID NO: 26 and 28 or SEQ ID NO:
2. An isolated nucleic acid molecule according to claim 1 containing a nucleotide sequence that can be amplified from a Lycopersicon esculentum DNA library using the polymerase chain reaction with a set of primers described as 59 and 60. 6. Beta vulgaris using the polymerase chain reaction with a set of primers set forth as SEQ ID NOs: 22 and 24 or SEQ ID NOs: 26 and 28.
2. The isolated nucleic acid molecule of claim 1, which contains a nucleotide sequence that can be amplified from a DNA library. 7. The isolated nucleic acid of claim 1, comprising a nucleotide sequence that can be amplified from a Helianthus annuus DNA library using the polymerase chain reaction with a set of primers set forth as SEQ ID NOs: 26 and 28. molecule. 8. A polymerase chain reaction with a set of primers set forth as SEQ ID NOs: 21 and 24, SEQ ID NOs: 21 and 23, SEQ ID NOs: 22 and 24, SEQ ID NOs: 25 and 28, or SEQ ID NOs: 26 and 28. Solanum tuberosum using
2. The isolated nucleic acid molecule of claim 1, which contains a nucleotide sequence that can be amplified from a DNA library. 9. Brassica napus using the polymerase chain reaction with a set of primers set forth as SEQ ID NOs: 9 and 10 or SEQ ID NOs: 26 and 28.
2. The isolated nucleic acid molecule of claim 1, which contains a nucleotide sequence that can be amplified from a DNA library. 10. A nucleotide sequence that can be amplified from an Arabidopsis thaliana DNA library using the polymerase chain reaction with a set of primers set forth as SEQ ID NOs: 13 and 14, SEQ ID NOs: 21 and 24 or SEQ ID NOs: 22 and 24. 2. The isolated nucleic acid molecule of claim 1, comprising: 11. SEQ ID NO: 9 and 10, SEQ ID NO: 11 and 12, SEQ ID NO:
21 and 24, SEQ ID NOs: 22 and 24, SEQ ID NOs: 25 and 28 or SEQ ID NO: 26
2. The isolated nucleic acid molecule of claim 1, comprising a nucleotide sequence that can be amplified from a Nicotiana tabacum DNA library using the polymerase chain reaction with a set of primers described as 28 and 28. 12. A set of primers corresponding to the first 20 nucleotides and SEQ ID NOs: 1, 3, 5, 7, 15, 17, 19, 29, 31, 33, 35, 37, 39, 41, 43, 45
, 47, 49, 51, 53, 55, 57, 61, 63, 65, 67, 69, 71 or 73 (C
DS) containing a nucleotide sequence that can be amplified from a plant DNA library using the polymerase chain reaction with the reverse complement of at least 20 nucleotides of
An isolated nucleic acid molecule according to claim 1. 13. A chimeric gene containing a promoter activity in a plant operably linked to the nucleic acid molecule according to claim 1. 14. A recombinant vector comprising the chimeric gene according to claim 13. 15. A host cell comprising the chimeric gene according to claim 13. 16. A plant comprising the chimeric gene according to claim 13. 17. The following plants: rice, wheat, barley, rye, corn, potato, canola, sunflower, carrot, sweet potato, sugar beet (sugar beet), kidney bean, peas, chicory, lettuce, cabbage, cauliflower, broccoli, Cabbage, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, pumpkin, pepo squash, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, 17. The plant of claim 16, wherein the plant is selected from blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum, and sugarcane. 18. A species from the plant according to claim 16. 19. A method for increasing SAR gene expression in a plant, comprising expressing the chimeric gene according to claim 13 in the plant. 20. A method for increasing disease resistance in a plant, comprising expressing the chimeric gene according to claim 13 in the plant. 21. A PCR primer selected from the group consisting of SEQ ID NOs: 9-14, 21-28, 59 and 60. 22. SEQ ID NO: 1, 3, 5, 7, 15, 17, 19, 29, 31, 33, 35, 3
7, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 61, 63, 65, 67, 69, 71 or 7
A set of primers corresponding to the reverse complement of the first 20 nucleotides and the last 20 nucleotides of the coding sequence (CDS) of SEQ ID NO: 3, or SEQ ID NO: 9 and 10, SEQ ID NO: 11 and 12, SEQ ID NO: : 13 and 14, SEQ ID NOs: 21 and 24, SEQ ID NOs: 22 and 24, SEQ ID NOs: 21 and 23, SEQ ID NOs: 25 and 28, SEQ ID NOs: 26 and 28 or one of SEQ ID NOs: 59 and 60 Isolate NIM1 homologues involved in signal transduction cascades leading to systemically acquired resistance in plants involving amplifying DNA molecules from a plant DNA library using the polymerase chain reaction with a set of primers Way for. 23. The plant DNA library is Nicotiana tabacum (tobacco).
), Lycopersicon esculentum (tomato), Brassica napus (oilseed), Arabido
psis thaliana, Beta vulgaris (sugar beet), Helianthus annuus (sunflower)
23. The method according to claim 22, wherein the method is a Solanum tuberosum (pot) DNA library.