Manipulation of Mlo genes to enhance disease resistance in plants

This application claims the benefit of U.S. Provisional Application No. 60/091,875, filed Jul. 7, 1998.

The invention relates to the genetic manipulation of plants, particularly to transforming plants with genes that enhance disease resistance.

Disease in plants is caused by biotic and abiotic causes. Biotic causes include fungi, viruses, bacteria, and nematodes. Of these, fungi are the most frequent causative agent of disease on plants. Abiotic causes of disease in plants include extremes of temperature, water, oxygen, soil pH, plus nutrient-element deficiencies and imbalances, excess heavy metals, and air pollution.

A host of cellular processes enables plants to defend themselves from disease caused by pathogenic agents. These processes apparently form an integrated set of resistance mechanisms that is activated by initial infection and then limits further spread of the invading pathogenic microorganism. This limitation of the pathogen intruder is frequently accomplished by localized containment of the intruder following a coordinated complex defense response.

Subsequent to recognition of a potentially pathogenic microbe, plants can activate an array of biochemical responses. Generally, the plant responds by inducing several local responses in the cells immediately surrounding the infection site. The most common resistance response observed in both nonhost and race-specific interactions is termed the “hypersensitive response” (HR). In the hypersensitive response, cells contacted by the pathogen, and often neighboring cells, rapidly collapse and dry in a necrotic fleck. Other responses include the deposition of callose, the physical thickening of cell walls by lignification, and the synthesis of various antibiotic small molecules and proteins. Genetic factors in both the host and the pathogen determine the specificity of these local responses, which can be very effective in limiting the spread of infection.

The hypersensitive response in many plant-pathogen interactions is specified by and dependent on the presence of two complementary genes, one from the host and one from the pathogen. These complementary genes are the resistance (R) gene in the plant and a corresponding avirulence (avr) gene in the pathogen. The interaction of the genes is associated with the rapid, localized cell death of the hypersensitive response. R genes that respond to specific bacterial, fungal, or viral pathogens, have been isolated from a variety of plant species and several appear to encode cytoplasmic proteins.

The resistance gene in the plant and the avirulence gene in the pathogen often conform to a gene-for-gene relationship. That is, resistance to a pathogen is only observed when the pathogen carries a specific avirulence gene and the plant carries a corresponding or complementing resistance gene. Because avr-R gene-for-gene relationships are observed in many plant-pathogen systems and are accompanied by a characteristic set of defense responses, a common molecular mechanism underlying avr-R gene mediated resistance has been postulated. A simple model which has been proposed is that pathogen avr genes directly or indirectly generate a specific molecular signal (ligand) that is recognized by cognate receptors encoded by plant R genes.

Both plant resistance genes and corresponding pathogen avirulence genes have been cloned. The plant kingdom contains thousands of R genes with specific specificities for viral, bacterial, fungal, or nematode pathogens. Although there are differences in the defense responses induced during different plant-pathogen interactions, some common themes are apparent among R gene-mediated defenses. The function of a given R gene is dependent on the genotype of the pathogen. Plant pathogens produce a diversity of potential signals, and in a fashion analogous to the production of antigens by mammalian pathogens, some of these signals are detectable by some plants.

The avirulence gene causes the pathogen to produce a signal that triggers a strong defense response in a plant with the appropriate R gene. However, expressing an avirulence gene does not stop the pathogen from being virulent on hosts that lack the corresponding R gene. A single plant can have many R genes, and a pathogen can have many avr genes.

Monogenic resistance mediated by recessive (mlo) alleles of the Mlo locus is different. It differs from race-specific incompatibility to single pathogen strains in that it is believed to confer a broad spectrum resistance to almost all known isolates of the fungal pathogen, and the resistance is apparently durable in the field despite extensive cultivation. Further, mlo resistance alleles have been obtained by mutagen treatment of susceptible wild-type Mlo varieties. These mlo plants exhibit a spontaneous leaf cell death phenotype under pathogen-free or even axenic conditions.

As noted, among the causative agents of infectious disease of crop plants, the phytopathogenic fungi play the dominant role. Phytopathogenic fungi cause devastating epidemics, as well as causing significant annual crop yield losses. All of the approximately 300,000 species of flowering plants are attacked by pathogenic fungi. However, a single plant species can be host to only a few fungal species, and similarly, most fungi usually have a limited host range.

Plant disease outbreaks have resulted in catastrophic crop failures that have triggered famines and caused major social change. Generally, the best strategy for plant disease control is to use resistant cultivars selected or developed by plant breeders for this purpose. However, the potential for serious crop disease epidemics persists today, as evidenced by outbreaks of the Victoria blight of oats and southern corn leaf blight.

Accordingly, molecular methods are needed to supplement traditional breeding methods to protect plants from pathogen attack. Particularly, methods are needed for broad spectrum resistance to pathogens.

Compositions and methods for creating or enhancing resistance to plant pests are provided. The method provides control of pathogens by modulating the expression of Mlo genes. Novel Mlo sequences are provided from maize. Such sequences can be utilized to modulate the expression of Mlo genes in plants, particularly maize, to enhance resistance to pathogens. Generally, such modulation will result in decreased or increased expression of native Mlo genes, preferably decreased expression. Such decreased expression can be effected by mutagenesis or expression of modified or antisense Mlo sequences described herein.

It is recognized that a variety of promoters will be useful in the invention the choice of which will depend in part upon the desired level of expression of the modified sequences in the plant or alternatively, in the plant organ. It is recognized that the levels of expression can be controlled to induce broad spectrum resistance resulting in levels of immunity in the plant or to induce cell death.

The methods of the invention find use in controlling plant pests, including fungal pathogens, viruses, nematodes, insects, and the like. Transformed plants and seeds, as well as methods for making such plants and seeds are additionally provided.

FIG. 1 provides a sequence alignment for the maize Mlo homologues with the barley Mlo sequence. The barley sequence is designated as HvMlo1.

FIG. 2 groups the Mlo sequences based on relatedness as well as the plant tissue from which it was isolated.

FIG. 3 schematically illustrates a plasmid vector comprising the ZmMlo 1 antisense construct operably linked to the ubiquition promoter.

Compositions of the invention include eight mutation-induced recessive alleles of maize set forth in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13 and 15. The maize sequences exhibit homology to the Mlo barley sequence (SEQ ID NO: 17). See, Büschges et al. (1997) 88:695-705 and Tables 1 and 2. The isolated maize Mlo genes are involved in enhancing resistance to plant pests. In particular, the present invention provides for isolated nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequences shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, or the nucleotide sequences encoding the DNA sequences deposited in a bacterial host with the American Type Culture Collection and assigned the Accession Nos. 98725, 98726, 98727, 98728, 98729 (SEQ ID NOS: 3, 5, 9, 11, 13 respectively). Further provided are polypeptides having an amino acid sequence encoded by a nucleic acid molecule described herein, for example those set forth in SEQ ID NOS: 1, 5, 3, 7, 9, 11, 13, and 15, those deposited with the American Type Culture Collection and assigned Accession Nos. 98725, 98726, 98727, 98728, 98729 (SEQ ID NOS: 3, 5, 9, 11, 13 respectively), and fragments and variants thereof.

Plasmids containing the nucleotide sequences of the invention were deposited with American Type Culture Collection, Manassas, Va., and assigned Accession Nos. 98725, 98726, 98727, 98728, 98729 (SEQ ID NOS: 3, 5, 9, 11, 13 respectively). These deposits will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. These deposits were made merely as a convenience for those of skill in the art and are not an admission that a deposit is required under 35 U.S.C. §112.

The invention encompasses isolated or substantially purified nucleic acid or protein compositions. An “isolated” or “purified” nucleic acid molecule or protein, or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Preferably, an “isolated” nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, (by dry weight) of contaminating protein. When the protein of the invention or biologically active portion thereof is recombinantly produced, preferably culture medium represents less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.

Fragments and variants of the disclosed nucleotide sequences and proteins encoded thereby are also encompassed by the present invention. By “fragment” is intended a portion of the nucleotide sequence or a portion of the amino acid sequence and hence protein encoded thereby. Fragments of a nucleotide sequence may encode protein fragments that retain the biological activity of the native protein and hence their altered expression enhances resistance to pathogens. Alternatively, fragments of a nucleotide sequence that are useful as hybridization probes generally do not encode protein fragments retaining biological activity. Thus, fragments of a nucleotide sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length nucleotide sequence encoding the proteins of the invention.

A fragment of a Mlo nucleotide sequence that encodes a biologically active portion of a Mlo protein of the invention will encode at least 15, 20, 25, 30, 40, 50, 75, 100, 200, 250, 300, 350, 400, 450, 500, or 550 contiguous amino acids, or up to the total number of amino acids present in a full-length MLO protein of the invention. (For example, 509, 499, and 492, amino acids for SEQ ID NOS: 6, 12, and 14 respectively.) Fragments of a Mlo nucleotide sequence that are useful as hybridization probes for PCR primers generally need not encode a biologically active portion of a MLO protein.

Thus, a fragment of a Mlo nucleotide sequence may encode a biologically active portion of a MLO protein, or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below. A biologically active portion of a MLO protein can be prepared by isolating a portion of one of the Mlo nucleotide sequences of the invention, expressing the encoded portion of the MLO protein (e.g., by recombinant expression in vitro), and assessing the activity of the encoded portion of the MLO protein. Nucleic acid molecules that are fragments of a Mlo nucleotide sequence comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, and 2,000 nucleotides, or up to the number of nucleotides present in a full-length Mlo nucleotide sequence disclosed herein.

By “variants” is intended substantially similar sequences. For nucleotide sequences, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the MLO polypeptides of the invention. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis but which still encode a MLO protein of the invention. Generally, nucleotide sequence variants of the invention will have at least 40%, 50%, 60%, 70%, generally, 80%, preferably 85%, 90%, up to 95%, 98% sequence identity to its respective native nucleotide sequence.

By “variant” protein is intended a protein derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Such variants may result from, for example, genetic polymorphism or from human manipulation.

The proteins of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the Mlo proteins can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol . 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be preferred.

Thus, the genes and nucleotide sequences of the invention include both the naturally occurring sequences as well as mutant forms. Likewise, the proteins of the invention encompass both naturally occurring proteins as well as variations and modified forms thereof. Such variants will continue to possess the desired ability to enhance resistance to pathogens when their expression is altered. Obviously, the mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. See, EP Patent Application Publication No. 75,444.

The deletions, insertions, and substitutions of the protein sequences encompassed herein are not expected to produce radical changes in the characteristics of the protein. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays. That is, the activity can be evaluated by an enhanced resistance to pathogens when the expression of the protein sequences is altered.

Variant nucleotide sequences and proteins also encompass sequences and proteins derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different Mlo coding sequences can be manipulated to create a new Mlo coding sequence possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest may be shuffled between the Mlo gene of the invention and other known genes involved in pathogen resistance to obtain a new gene coding for a protein with an improved property of interest, such as an increased K m in the case of an enzyme. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech . 15:436-438; Moore et al. (1997) J. Mol. Biol . 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

The maize Mlo homologues are associated with disease related loci as shown in Table 3. Mapping information of the sequences are given in Table 5. The maize homologue proteins are predicted to be membrane-anchored by at least six, and possibly seven membrane-spanning helices. The maize sequences find use in negative control function of the MLO protein in leaf cell death and in the onset of pathogen defense. Generally, the methods of the invention take advantage of the absence of MLO to prime responsiveness of the plant to disease.

The nucleotide sequences of the invention can be used to isolate corresponding sequences from other organisms, particularly other plants, more particularly other monocots. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences set forth herein. Sequences isolated based on their sequence identity to the entire Mlo sequences set forth herein or to fragments thereof are encompassed by the present invention.

In a PCR approach, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any plant of interest. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially-mismatched primers, and the like.

In hybridization techniques, all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as 32 P, or any other detectable marker. Thus, for example, probes for hybridization can be made by labeling synthetic oligonucleotides based on the Mlo sequences of the invention. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

For example, the entire sequence of the Mlo nucleotide sequences disclosed herein, or one or more portions thereof, may be used as a probe capable of specifically hybridizing to corresponding Mlo sequences and messenger RNAs. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique among Mlo sequences and are preferably at least about 10 nucleotides in length, and most preferably at least about 20 nucleotides in length. Such probes may be used to amplify corresponding Mlo sequences from a chosen plant by PCR. This technique may be used to isolate additional coding sequences from a desired plant or as a diagnostic assay to determine the presence of coding sequences in a plant. Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

Hybridization of such sequences may be carried out under stringent conditions. By “stringent conditions” or “stringent hybridization conditions” is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C.

Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the T m can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem . 138:267-284: T m =81.5° C.+16.6 (log M)+0.41 (% GC) −0.61 (% form) −500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The T m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. T m is reduced by about 1° C. for each 1% of mismatching; thus, T m , hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with ≧90% identity are sought, the T m can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (T m ) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermal melting point (T m ); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than the thermal melting point (T m ); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the thermal melting point (T m ). Using the equation, hybridization and wash compositions, and desired T m , those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a T m of less than 45° C. (aqueous solution) or 32° C. (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes , Part I, Chapter 2 (Elsevier, N.Y.); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology , Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

In general, sequences that encode for a Mlo protein and hybridize to the Mlo sequences disclosed herein will be at least 40% to 50% homologous, about 60% to 70% homologous, and even about 80%, 85%, 90%, 95% to 98% homologous or more with the disclosed sequences. That is, the sequence similarity of sequences may range, sharing at least about 40% to 50%, about 60% to 70%, and even about 80%, 85%, 90%, 95% to 98% sequence similarity.

The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) “reference sequence”, (b) “comparison window”, (c) “sequence identity”, (d) “percentage of sequence identity”, and (e) “substantial identity”.

(a) As used herein, “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.

(b) As used herein, “comparison window” makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith et al. (1981) Adv. AppL. Math . 2:482; by the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol . 48:443; by the search for similarity method of Pearson et al. (1988) Proc. Natl. Acad. Sci . 85:2444; by computerized implementations of these algorithms, including, but not limited to: CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, Calif.; GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA; the CLUSTAL program is well described by Higgins et al. (1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153; Corpet et al. (1988) Nucleic Acids Res . 16:10881-90; Huang et al. (1992) Computer Applications in the Biosciences 8:155-65, and Person et al. (1994) Meth. Mol. Biol . 24:307-331; preferred computer alignment methods also include the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol . 215:403-410). Alignment is also often performed by inspection and manual alignment.

(c) As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity”. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

(e)(i) The term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70% sequence identity, preferably at least 80%, more preferably at least 90%, and most preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 60%, more preferably at least 70%, 80%, 90%, and most preferably at least 95%.

Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH. However, stringent conditions encompass temperatures in the range of about 1° C. to about 20° C., depending upon the desired degree of stringency as otherwise qualified herein. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.

(e)(ii) The term “substantial identity” in the context of a peptide indicates that a peptide comprises a sequence with at least 70% sequence identity to a reference sequence, preferably 80%, more preferably 85%, most preferably at least 90% or 95% sequence identity to the reference sequence over a specified comparison window. Preferably, optimal alignment is conducted using the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol . 48:443. An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution. Peptides that are “substantially similar” share sequences as noted above except that residue positions that are not identical may differ by conservative amino acid changes.

The invention is drawn to methods for creating or enhancing resistance in a plant to plant pests by modulating the activity of Mlo genes in the plant. While the invention is not bound by any particular mechanism of action, it is believed that the methods of the invention will result in broad-based resistance in the modified plant. Accordingly, the methods are also useful in protecting plants against fungal pathogens, viruses, nematodes, insects and the like.

By “modulating activity” is intended that the expression of the Mlo gene is altered in some manner. Such modulation (increase or decrease) of expression results in enhanced resistance to pathogens. Generally, the methods of the invention will result in a decrease in the native protein or in protein activity. Thus, plants and plant cells are obtained having altered levels of MLO protein, preferably a decrease in protein levels. Such plants, plant cells and plant tissues are “modified” in that MLO protein levels are altered. As noted below, various methods are available for creating modified plants, plant cells and plant tissues including transformation and transfection leading to altered Mlo expression in the modified plant, plant cell or tissue.

The invention is drawn to compositions and methods for inducing resistance in a plant to plant pests. Accordingly, the compositions and methods are also useful in protecting plants against fungal pathogens, viruses, nematodes, insects and the like.

By “disease resistance” is intended that the plants avoid the disease symptoms that are the outcome of plant-pathogen interactions. That is, pathogens are prevented from causing plant diseases and the associated disease symptoms, or alternatively, the disease symptoms caused by the pathogen is minimized or lessened. The methods of the invention can be utilized to protect plants from disease, particularly those diseases that are caused by plant pathogens.

Pathogens of the invention include, but are not limited to, viruses or viroids, bacteria, insects, nematodes, fungi, and the like. Viruses include any plant virus, for example, tobacco or cucumber mosaic virus, ringspot virus, necrosis virus, maize dwarf mosaic virus, etc. Specific fungal and viral pathogens for the major crops include: Soybeans: Phytophthora megasperma fsp. glycinea, Macrophomina phaseolina, Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium oxysporum, Diaporthe phaseolorum var. sojae ( Phomopsis sojae ), Diaporthe phaseolorum var. caulivora, Sclerotium rolfsii, Cercospora kikuchii, Cercospora sojina, Peronospora manshurica, Colletotrichum dematium ( Colletotichum truncatum ), Corynespora cassiicola, Septoria glycines, Phyllosticta sojicola, Alternaria alternata, Pseudomonas syringae p.v. glycinea, Xanthomonas campestris p.v. phaseoli, Microsphaera diffusa, Fusarium semitectum, Phialophora gregata, Soybean mosaic virus, Glomerella glycines , Tobacco Ring spot virus, Tobacco Streak virus, Phakopsora pachyrhizi, Pythium aphanidermatum, Pythium ultimum, Pythium debaryanum , Tomato spotted wilt virus, Heterodera glycines Fusarium solani ; Canola: Albugo candida, Alternaria brassicae, Leptosphaeria maculans, Rhizoctonia solani, Sclerotinia sclerotiorum, Mycosphaerella brassiccola, Pythium ultimum, Peronospora parasitica, Fusarium roseum, Alternaria alternata ; Alfalfa: Clavibater michiganese subsp. insidiosum, Pythium ultimum, Pythium irregulare, Pythium splendens, Pythium debaryanum, Pythium aphanidermatum, Phytophthora megasperma, Peronospora trifoliorum, Phoma medicaginis var. medicaginis, Cercospora medicaginis, Pseudopeziza medicaginis, Leptotrochila medicaginis, Fusarium, Xanthomonas campestris p.v. alfalfae, Aphanomyces euteiches, Stemphylium herbarum, Stemphylium alfalfae ; Wheat: Pseudomonas syringae p.v. atrofaciens, Urocystis agropyri, Xanthomonas campestris p.v. translucens, Pseudomonas syringae p.v. syringae, Alternaria alternata, Cladosporium herbarum, Fusarium graminearum, Fusarium avenaceum, Fusarium culmorum, Ustilago tritici, Ascochyta tritici, Cephalosporium gramineum, Collotetrichum graminicola, Erysiphe graminis f.sp. tritici, Puccinia graminis f.sp. tritici, Puccinia recondita f.sp. tritici, Puccinia striiformis, Pyrenophora tritici-repentis, Septoria nodorum, Septoria tritici, Septoria avenae, Pseudocercosporella herpotrichoides, Rhizoctonia solani, Rhizoctonia cerealis, Gaeumannomyces graminis var. tritici, Pythium aphanidermatum, Pythium arrhenomanes, Pythium ultimum, Bipolaris sorokiniana , Barley Yellow Dwarf Virus, Brome Mosaic Virus, Soil Borne Wheat Mosaic Virus, Wheat Streak Mosaic Virus, Wheat Spindle Streak Virus, American Wheat Striate Virus, Claviceps purpurea, Tilletia tritici, Tilletia laevis, Ustilago tritici, Tilletia indica, Rhizoctonia solani, Pythium arrhenomannes, Pythium gramicola, Pythium aphanidermatum , High Plains Virus, European wheat striate virus; Sunflower: Plasmophora halstedii, Sclerotinia sclerotiorum , Aster Yellows, Septoria helianthi, Phomopsis helianthi, Alternaria helianthi, Alternaria zinniae, Botrytis cinerea, Phoma macdonaldii, Macrophomina phaseolina, Erysiphe cichoracearum, Rhizopus oryzae, Rhizopus arrhizus, Rhizopus stolonifer, Puccinia helianthi, Verticillium dahliae, Erwinia carotovorum pv. carotovora, Cephalosporium acremonium, Phytophthora cryptogea, Albugo tragopogonis ; Corn: Fusarium moniliforme var. subglutinans, Erwinia stewartii, Fusarium moniliforme, Gibberella zeae (Fusarium graminearum), Stenocarpella maydi (Diplodia maydis), Pythium irregulare, Pythium debaryanum, Pythium graminicola, Pythium splendens, Pythium ultimum, Pythium aphanidermatum, Aspergillus flavus, Bipolaris maydis O, T ( Cochliobolus heterostrophus ), Helminthosporium carbonum I, II & III ( Cochliobolus carbonum ), Exserohilum turcicum I, II & III, Helminthosporium pedicellatum, Physoderma maydis, Phyllosticta maydis, Kabatiella-maydis, Cercospora sorghi, Ustilago maydis, Puccinia sorghi, Puccinia polysora, Macrophomina phaseolina, Penicillium oxalicum, Nigrospora oryzae, Cladosporium herbarum, Curvularia lunata, Curvularia inaequalis, Curvularia pallescens, Clavibacter michiganense subsp. nebraskense, Trichoderma viride , Maize Dwarf Mosaic Virus A & B, Wheat Streak Mosaic Virus, Maize Chlorotic Dwarf Virus, Claviceps sorghi, Pseudonomas avenae, Erwinia chrysanthemi pv. zea, Erwinia carotovora, Corn stunt spiroplasma, Diplodia macrospora, Sclerophthora macrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis, Peronosclerospora maydis, Peronosclerospora sacchari, Sphacelotheca reiliana, Physopella zeae, Cephalosporium maydis, Cephalosporium acremonium , Maize Chlorotic Mottle Virus, High Plains Virus, Maize Mosaic Virus, Maize Rayado Fino Virus, Maize Streak Virus, Maize Stripe Virus, Maize Rough Dwarf Virus; Sorghum: Exserohilum turcicum, Colletotrichum graminicola ( Glomerella graminicola ), Cercospora sorghi, Gloeocercospora sorghi, Ascochyta sorghina, Pseudomonas syringae p.v. syringae, Xanthomonas campestris p.v. holcicola, Pseudomonas andropogonis, Puccinia purpurea, Macrophomina phaseolina, Perconia circinata, Fusarium moniliforme, Alternaria alternata, Bipolaris sorghicola, Helminthosporium sorghicola, Curvularia lunata, Phoma insidiosa, Pseudomonas avenae ( Pseudomonas alboprecipitans ), Ramulispora sorghi, Ramulispora sorghicola, Phyllachara sacchari, Sporisorium reilianum ( Sphacelotheca reiliana ), Sphacelotheca cruenta, Sporisorium sorghi, Sugarcane mosaic H, Maize Dwarf Mosaic Virus A & B, Claviceps sorghi, Rhizoctonia solani, Acremonium strictum, Sclerophthona macrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis, Sclerospora graminicola, Fusarium graminearum, Fusarium oxysporum, Pythium arrhenomanes, Pythium graminicola , etc.

Nematodes include parasitic nematodes such as root-knot, cyst, lesion, and renniform nematodes, etc.

Insect pests include insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Coleoptera and Lepidoptera. Insect pests of the invention for the major crops include: Maize: Ostrinia nubilalis , European corn borer; Agrotis ipsilon , black cutworm; Helicoverpa zea, corn earworm; Spodoptera frugiperda , fall armyworm; Diatraea grandiosella , southwestern corn borer; Elasmopalpus lignosellus , lesser cornstalk borer; Diatraea saccharalis , surgarcane borer; Diabrotica virgifera , western corn rootworm; Diabrotica longicornis barberi , northern corn rootworm; Diabrotica undecimpunctata howardi , southern corn rootworm; Melanotus spp., wireworms; Cyclocephala borealis , northern masked chafer (white grub); Cyclocephala immaculata , southern masked chafer (white grub); Popillia japonica , Japanese beetle; Chaetocnema pulicaria , corn flea beetle; Sphenophorus maidis , maize billbug; Rhopalosiphum maidis , corn leaf aphid; Anuraphis maidiradicis , corn root aphid; Blissus leucopterus leucopterus , chinch bug; Melanoplus femurrubrum , redlegged grasshopper; Melanoplus sanguinipes , migratory grasshopper; Hylemya platura , seedcorn maggot; Agromyza parvicornis , corn blot leafminer; Anaphothrips obscrurus , grass thrips; Solenopsis milesta , thief ant; Tetranychus urticae , twospotted spider mite; Sorghum: Chilo partellus , sorghum borer; Spodoptera frugiperda , fall armyworm; Helicoverpa zea, corn earworm; Elasmopalpus lignosellus , lesser cornstalk borer; Feltia subterranea , granulate cutworm; Phyllophaga crinita , white grub; Eleodes, Conoderus, and Aeolus spp., wireworms; Oulema melanopus , cereal leaf beetle; Chaetocnema pulicaria , corn flea beetle; Sphenophorus maidis , maize billbug; Rhopalosiphum maidis ; corn leaf aphid; Sipha flava , yellow sugarcane aphid; Blissus leucopterus leucopterus , chinch bug; Contarinia sorghicola , sorghum midge; Tetranychus cinnabarinus , carmine spider mite; Tetranychus urticae , twospotted spider mite; Wheat: Pseudaletia unipunctata , army worm; Spodoptera frugiperda , fall armyworm; Elasmopalpus lignosellus , lesser cornstalk borer; Agrotis orthogonia , western cutworm; Elasmopalpus lignosellus , lesser cornstalk borer; Oulema melanopus , cereal leaf beetle; Hypera punctata , clover leaf weevil; Diabrotica undecimpunctata howardi , southern corn rootworm; Russian wheat aphid; Schizaphis graminum , greenbug; Macrosiphum avenae , English grain aphid; Melanoplus femurrubrum , redlegged grasshopper; Melanoplus differentialis , differential grasshopper; Melanoplus sanguinipes , migratory grasshopper; Mayetiola destructor , Hessian fly; Sitodiplosis mosellana , wheat midge; Meromyza americana , wheat stem maggot; Hylemya coarctata , wheat bulb fly; Frankliniella fusca , tobacco thrips; Cephus cinctus , wheat stem sawfly; Aceria tulipae , wheat curl mite; Sunflower: Suleima helianthana , sunflower bud moth; Homoeosoma electellum , sunflower moth; zygogramma exclamationis , sunflower beetle; Bothyrus gibbosus , carrot beetle; Neolasioptera murtfeldtiana , sunflower seed midge; Cotton: Heliothis virescens , cotton budworm; Helicoverpa zea, cotton bollworm; Spodoptera exigua , beet armyworm; Pectinophora gossypiella , pink bollworm; Anthonomus grandis grandis , boll weevil; Aphis gossypii , cotton aphid; Pseudatomoscelis seriatus , cotton fleahopper; Trialeurodes abutilonea , bandedwinged whitefly; Lygus lineolaris , tarnished plant bug; Melanoplus femurrubrum , redlegged grasshopper; Melanoplus differentialis , differential grasshopper; Thrips tabaci , onion thrips; Franklinkiella fusca , tobacco thrips; Tetranychus cinnabarinus , carmine spider mite; Tetranychus urticae , twospotted spider mite; Rice: Diatraea saccharalis , sugarcane borer; Spodoptera frugiperda , fall armyworm; Helicoverpa zea, corn earworm; Colaspis brunnea , grape colaspis; Lissorhoptrus oryzophilus , rice water weevil; Sitophilus oryzae , rice weevil; Nephotettix nigropictus , rice leafhopper; Blissus leucopterus leucopterus , chinch bug; Acrosternum hilare , green stink bug; Soybean: Pseudoplusia includens , soybean looper; Anticarsia gemmatalis , velvetbean caterpillar; Plathypena scabra , green cloverworm; Ostrinia nubilalis , European corn borer; Agrotis ipsilon , black cutworm; Spodoptera exigua , beet armyworm; Heliothis virescens , cotton budworm; Helicoverpa zea, cotton bollworm; Epilachna varivestis , Mexican bean beetle; Myzus persicae , green peach aphid; Empoasca fabae , potato leafhopper; Acrosternum hilare , green stink bug; Melanoplus femurrubrum , redlegged grasshopper; Melanoplus differentialis , differential grasshopper; Hylemya platura , seedcorn maggot; Sericothrips variabilis , soybean thrips; Thrips tabaci , onion thrips; Tetranychus turkestani , strawberry spider mite; Tetranychus urticae , twospotted spider mite; Barley: Ostrinia nubilalis , European corn borer; Agrotis epsilon , black cutworm; Schizaphis graminum , greenbug; Blissus leucopterus leucopterus , chinch bug; Acrosternum hilare , green stink bug; Euschistus servus , brown stink bug; Delia platura , seedcorn maggot; Mayetiola destructor , Hessian fly; Petrobia latens , brown wheat mite; Oil Seed Rape: Brevicoryne brassicae , cabbage aphid; Phyllotreta cruciferae , Flea beetle; Mamestra configurata , Bertha armyworm; Plutella xylostella , Diamond-back moth; Delia ssp., Root maggots.

The present invention exploits the use of Mlo genes. The Mlo sequences provided herein can be utilized to alter the expression of native Mlo genes in plants. The mlo mutation confers recessive resistance to pathogens. Broad spectrum resistance in plants can be enhanced by defective Mlo genes.

While the invention is not bound by any model, Mlo could have a negative control function in leaf cell death. In this model, Mlo would suppress a default cell suicide program in foliar tissue. Also, the MLO protein could have a specific negative regulatory function which works by down-regulating multiple disease-related functions. In this instance, spontaneous cell death in mlo mutant genotypes merely represents cell death because of accumulating activation of defense responses.

Several methods are available in the art for modulating the activity of Mlo genes. Mlo antisense sequences can be expressed in the plant cell. Such sequences will function to decrease expression of the maize Mlo genes as well as Mlo genes in other plants where the Mlo sequences share sequence identity.

It is recognized that with these nucleotide sequences, antisense constructions, complementary to at least a portion of the messenger RNA (mRNA) for the Mlo sequences can be constructed. Antisense nucleotides are constructed to hybridize with the corresponding mRNA. Modifications of the antisense sequences may be made as long as the sequences hybridize to and interfere with expression of the corresponding mRNA. In this manner, antisense constructions having 70%, preferably 80%, more preferably 85% sequence similarity to the corresponding antisensed sequences may be used. Furthermore, portions of the antisense nucleotides may be used to disrupt the expression of the target gene. Generally, sequences of at least 50 nucleotides, 100 nucleotides, 200 nucleotides, or greater may be used.

The antisense construct may be proximal to the 5′-terminus or capping site, downstream from the capping site, between the capping site and the initiation codon and may cover all or a portion of the non-coding region. Additionally, the sequence may bridge the non-coding and coding region, be complementary to all or part of the coding region, to the 3′-terminus of the coding region, or complementary to the 3′-untranslated region of the mRNA.

It is recognized that the particular site to which the antisense sequence binds and the length of the antisense sequence will vary depending upon the degree of inhibition desired, the uniqueness of the sequence, the stability of the antisense sequence, and the like. See, for example, U.S. Pat. Nos. 5,453,566; 5,530,192; and 5,728,926; all of which are herein incorporated by reference.

The nucleotide sequences of the present invention may also be used in the sense orientation to suppress the expression of endogenous genes in plants. Methods for suppressing gene expression in plants using nucleotide sequences in the sense orientation are known in the art. The methods generally involve transforming plants with a DNA construct comprising a promoter that drives expression in a plant operably linked to at least a portion of a nucleotide sequence that corresponds to the transcript of the endogenous gene. Typically, such a nucleotide sequence has substantial sequence identity to the sequence of the transcript of the endogenous gene, preferably greater than about 65% sequence identity, more preferably greater than about 85% sequence identity, most preferably greater than about 95% sequence identity. See, U.S. Pat. Nos. 5,283,184 and 5,034,323; herein incorporated by reference.

Individual Mlo genes or sets of Mlo genes can be rendered nonfunctional by mutagenesis. Such mutagenesis techniques include transposon disruption and recovery of such disruptions by reverse genetics approaches. Likewise, transformation-mediated mutagenesis may be utilized.

Disruption may be accomplished by transformation with gene replacement or gene truncation-disruption. See, for example, Bowen et al. (1995) Mol. Gen. Gent . 246:196-205; Walz et al. (1993) Curr. Gent . 25:421-427; Sweigard et al. (1992) Mol. Gen. Gent . 232:183-190; Hohn and Desjardins (1992) Mol. Plant Microbe Interact 5:249-256; Weber and Laitner (1994) Curr. Gent . 26:461-467; Templeton et al. (1994) Gene 142:141-146; Gorlach et al. (1998) Appl. Environ. Microbiol . 64:385-391; Schaeffer et al. (1994) Appl. Environ. Microbiol . 60:594-598; and the like, herein incorporated by reference. See also, Kempin et al. (1997) Nature 389:802-803 and Koncz et al. (1992) Plant Mol. Biol . 20:963-976.

Alternatively, the naturally occurring Mlo sequence or sequences may be modified by site-directed mutagenesis. Such methods may be utilized to induce specific alterations in targeted genes. One means for site-directed mutagenesis includes targeting modification or mutation of the Mlo sequences by homologous recombination. The method involves the use of RNA-DNA hybrid oligonucleotides. Such nucleotides exploit the natural recombinogenicity of RNA-DNA hybrids. The oligonucleotides are duplex oligonucleotides that share homology with at least one Mlo sequence. While any region of the Mlo sequence can be targeted, it may be preferable to target the 5′ region of the Mlo sequence. See, for example, U.S. Pat. No, 5,565,350 that describes chimeric oligonucleotides useful for targeted gene correction for use in cultured mammalian cells; as well as U.S. provisional application Serial No. 06/065,628 drawn to gene manipulation in plant cells, herein incorporated by reference. Such methods can be used to alter or disrupt the ATG start codon for the gene.

Alternatively, protein coding regions of Mlo genes can be altered in such a manner that the gene products or proteins perform their function in a dominant negative manner resulting in a resistant phenotype. See, for example Krylov et al. (1997) Proc. Natl. Acad. Sci. USA 94:12274-12279.

Where the method of the invention relies upon the expression of an altered Mlo sequence or an Mlo antisense sequence in a plant, a number of promoters can be used. The promoters can be selected from constitutive and/or inducible promoters. Such promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen; e.g., PR proteins, SAR proteins, beta-1,3-glucanase, chitinase, etc. See, for example, Redolfi et al. (1983) Neth. J. Plant Pathol . 89:245-254; Uknes et al. (1992) The Plant Cell 4:645-656; and Van Loon (1985) Plant Mol. Virol . 4:111-116. See, also copending application entitled “Inducible Maize promoters”, filed and herein incorporated by reference.

Of interest are promoters that are expressed locally at or near the site of pathogen infection. See, for example, Marineau et al. (1987) Plant Mol. Biol . 9:335-342; Matton et al. (1989) Molecular Plant-Microbe Interactions 2:325-331; Somsisch et al. (1986) Proc. Natl. Acad. Sci. USA 83:2427-2430; Somsisch et al. (1988) Molecular and General Genetics 2:93-98; and Yang, Y (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen et al. (1996) Plant J . 10:955-966; Zhang and Sing (1994) Proc. Natl Acad. Sci. USA 91:2507-2511; Warner et al. (1993) Plant J . 3:191-201; Siebertz et al. (1989) Plant Cell 1:961-968; and the references cited therein. Of particular interest is the inducible promoter for the maize PRms gene, whose expression is induced by the pathogen Fusarium moniliforme (see, for example, Cordero et al. (1992) Physiological and Molecular Plant Pathology 41:189-200).

Constitutive promoters include, for example, the core promoter of the Rsyn7 (copending application Ser. No. 08/661,601), the 35S promoter, the core 35S promoter, and the like. Other constitutive promoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142. See also, copending application entitled “Constitutive Maize Promoters” Provisional Application Serial No. 60/076,075 filed Feb. 26, 1998, and herein incorporated by reference.

Tissue specific promoters include Yamamoto et al. (1997) Plant J . 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol . 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet . 254(3):337-343; Russell et al. (1997) Transgenic Res . 6(2):157-168; Rinehart et al. (1996) Plant Physiol . 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol . 112(2):525-535; Canevascini et al. (1996) Plant Physiol . 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol . 35(5):773-778; Lam (1994) Results Probl. Cell. Differ . 20:181-196; Orozco et al. (1993) Plant Mol. Biol . 23(6): 1129-1138; Matsuoka et al. (1993) Proc. Natl. Acad. Sci. U.S.A . 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J . 4(3):495-505.

The methods of the invention can be used with other methods for increasing pathogen resistance in plants. See, for example, Cai et al. (1997) Science 275:832-834; Roberts and Gallum (1984) J. Heredity 75:147-148; Ryerson and Heath (1996) Plant Cell 8:393-402 and Dangl et al. (1996) Plant Cell 8:1793-1807.

Altered Mlo sequences or antisense Mlo sequences of the invention can be introduced into any plant. The sequences to be introduced may be used in expression cassettes for expression in any plant of interest where expression in the plant cell is necessary. In other instances, such as for recombination, oligonucleotides are synthesized, purified and introduced into the plant cell.

Where expression cassettes are needed, such expression cassettes will comprise a transcriptional initiation region linked to the coding sequence or antisense sequence of the nucleotide of interest. Such an expression cassette is provided with a plurality of restriction sites for insertion of the sequence to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.

The transcriptional initiation region, the promoter, may be native or analogous or foreign or heterologous to the plant host. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. By foreign is intended that the transcriptional initiation region is not found in the native plant into which the transcriptional initiation region is introduced. As used herein a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.

While it may be preferable to express the sequences using heterologous promoters, the native promoter sequences may be used. Such constructs would change expression levels of a MLO protein in the plant or plant cell. Thus, the phenotype of the plant or plant cell is altered.

The transcriptional cassette will include in the 5′-3′ direction of transcription, a transcriptional and translational initiation region, a DNA sequence of interest, and a transcriptional and translational termination region functional in plants. The termination region may be native with the transcriptional initiation region, may be native with the DNA sequence of interest, or may be derived from another source. Convenient termination regions are available from the Ti-plasmid of A. tumefaciens , such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau et al. (1991) Mol. Gen. Genet . 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev . 5:141-149; Mogen et al. (1990) Plant Cell . 2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res . 17:7891-7903; Joshi et al. (1987) Nucleic Acid Res . 15:9627-9639.

Nucleotide sequences of the invention are provided in expression cassettes for expression in the plant of interest. The cassette will include 5′ and 3′ regulatory sequences operably linked to the sequence of interest. The cassette may additionally contain at least one additional sequence to be cotransformed into the organism. Alternatively, the additional sequence(s) can be provided on another expression cassette.

Where appropriate, the gene(s) may be optimized for increased expression in the transformed plant. That is, the genes can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri (1990) Plant Physiol . 92:1-11 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831, 5,436,391, and Murray et al. (1989) Nucleic Acids Res . 17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.

The expression cassettes may additionally contain 5′ leader sequences in the expression cassette construct. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al. (1989) PNAS USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Allison et al. (1986); MDMV leader (Maize Dwarf Mosaic Virus); Virology 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP), (Macejak et al. (1991) Nature 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, N.Y.), pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991) Virology 81:382-385). See also, Della-Cioppa et al. (1987) Plant Physiol . 84:965-968. Other methods known to enhance translation can also be utilized, for example, introns, and the like.

In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Towards this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g. transitions and transversions, may be involved.

The sequences of the present invention can be used to transform or transfect any plant. In this manner, genetically modified plants, plant cells, plant tissue, seed, and the like can be obtained. Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediated transformation (Townsend et al., U.S. Pat No. 5,563,055), direct gene transfer (Paszkowski et al. (1984) EMBO J . 3:2717-2722), and ballistic particle acceleration (see, for example, Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al. (1995) “Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture: Fundamental Methods , ed. Gamborg and Phillips (Springer-Verlag, Berlin); and McCabe et al. (1988) Biotechnology 6:923-926). Also see Weissinger et al. (1988) Ann. Rev. Genet . 22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol . 87:671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol . 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet . 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad Sci. USA 85:43054309 (maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); Tomes, U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos. 5,322,783 and 5,324,646; Tomes et al. (1995) “Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture: Fundamental Methods , ed. Gamborg (Springer-Verlag, Berlin) (maize); Klein et al. (1988) Plant Physiol . 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311:763-764; Bowen et al., U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues , ed. Chapman et al. (Longman, N.Y.), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet . 84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference.

The modified plant may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell. Reports , 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that the subject phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure the desired phenotype or other property has been achieved.

The Mlo sequences of the invention are also useful as molecular markers. Such markers are useful in breeding programs, particularly those aimed at improving disease resistance. The maize Mlo sequences have been mapped to chromosome locations and these positions related to known disease resistance loci.

The present invention provides a method of genotyping a plant comprising a polynucleotide of the present invention. Preferably, the plant is a monocot, such as maize or sorghum. Genotyping provides a means of distinguishing homologs of a chromosome pair and can be used to differentiate segregants in a plant population. Molecular marker methods can be used for phylogenetic studies, characterizing genetic relationships among crop varieties, identifying crosses or somatic hybrids, localizing chromosomal segments affecting monogenic traits, map based cloning, and the study of quantitative inheritance. See, e.g., Plant Molecular Biology: A Laboratory Manual , Chapter 7, Clark, Ed., Springer-Verlag, Berlin (1997). For molecular marker methods, see generally, The DNA Revolution by Andrew H. Paterson (1996) (Chapter 2) in: Genome Mapping in Plants (ed. Andrew H. Paterson) by Academic Press/R. G. Landis Company, Austin, Texas, pp. 7-21.

The particular method of genotyping in the present invention may employ any number of molecular marker analytic techniques such as, but not limited to, restriction fragment length polymorphisms (RFLPs). RFLPs are the product of allelic differences between DNA restriction fragments caused by nucleotide sequence variability. As is well known to those of skill in the art, RFLPs are typically detected by extraction of genomic DNA and digestion with a restriction enzyme. Generally, the resulting fragments are separated according to size and hybridized with a probe; single copy probes are preferred. Restriction fragments from homologous chromosomes are revealed. Differences in fragment size among alleles represent an RFLP. Thus, the present invention further provides a means to follow segregation of a gene or nucleic acid of the present invention as well as chromosomal sequences genetically linked to these genes or nucleic acids using such techniques as RFLP analysis. Linked chromosomal sequences are within 50 centiMorgans (Cm), often within 40 or 30 Cm, preferably within 20 or 10 Cm, more preferably within 5, 3, 2, or 1 Cm of a gene.

In the present invention, the nucleic acid probes employed for molecular marker mapping of plant nuclear genomes selectively hybridize, under selective hybridization conditions, to a gene encoding a polynucleotide of the present invention. In preferred embodiments, the probes are selected from polynucleotides of the present invention. Typically, these probes are cDNA probes or PstI genomic clones. The length of the probes is discussed in greater detail, supra, but are typically at least 15 bases in length, more preferably at least 20, 25, 30, 35, 40, or 50 bases in length. Generally, however, the probes are less than about 1 kilobase in length. Preferably, the probes are single copy probes that hybridize to a unique locus in a haploid chromosome complement. Some exemplary restriction enzymes employed in RFLP mapping are EcoRI, EcoRv, and SstI. As used herein the term “restriction enzyme” includes reference to a composition that recognizes and, alone or in conjunction with another composition, cleaves at a specific nucleotide sequence.

The method of detecting an RFLP comprises the steps of (a) digesting genomic DNA of a plant with a restriction enzyme; (b) hybridizing a nucleic acid probe, under selective hybridization conditions, to a sequence of a polynucleotide of the present of said genomic DNA; (c) detecting therefrom a RFLP. Other methods of differentiating polymorphic (allelic) variants of polynucleotides of the present invention can be had by utilizing molecular marker techniques well known to those of skill in the art including such techniques as: 1) single-stranded conformation analysis (SSCP); 2) denaturing gradient gel electrophoresis (DGGE); 3) Rnase protection assays; 4) allele-specific oligonucleotides (ASOs); 5) the use of proteins which recognize nucleotide mismatches, such as the E. coli mutS protein; and 6) allele-specific PCR. Other approaches based on the detection of mismatches between the two complementary DNA strands include clamped denaturing gel electrophoresis (CDGE); heteroduplex analysis (HA); and chemical mismatch cleavage (CMC). Exemplary polymorphic variants are provided in Table I, supra. Thus, the present invention further provides a method of genotyping comprising the steps of contacting, under stringent hybridization conditions, a sample suspected of comprising a polynucleotide of the present invention with a nucleic acid probe. Generally, the sample is a plant sample; preferably, a sample suspected of comprising a maize polynucleotide of the present invention (e.g., gene, mRNA). The nucleic acid probe selectively hybridizes, under stringent conditions, to a subsequence of a polynucleotide of the present invention comprising a polymorphic marker. Selective hybridization of the nucleic acid probe to the polymorphic marker nucleic acid sequence yields a hybridization complex. Detection of the hybridization complex indicates the presence of that polymorphic marker in the sample. In preferred embodiments, the nucleic acid probe comprises a polynucleotide of the present invention.

The following examples are offered by way of illustration and not by way of limitation.

Mlo Sequences

Eight maize Mlo homologue sequences have been identified. The nucleotide sequences (SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, and 15) and amino acid sequences (SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, and 16) are provided. DNA sequence analysis was performed according to Sambrook et al. (1989) Mol. Cloning: A Laboratory Manual , 2nd ed. (Cold Spring Harbor Laboratory Press, Plainview, N.Y.). The maize Mlo homologue/orthologue cDNA sequences were identified from mRNA isolated from various maize tissues and used to make cDNAs that are then cloned into vectors, usually pSPORT1. These cDNA libraries are EST sequenced via standard dye-fluorescence labeling and ABI machine electrophoresis and image capture. The Mlo homologues were identified by their blast score identity to the barley HvMLO1 published sequence. These maize clones, in particular the longest members of each contig or gene, were obtained and additional sequencing was done on them using oligonucleotide primers designed to internal portions of the cDNA and dye-fluorescence labeling and ABI machine electrophoresis and image capture. The complete edited sequences were assembled and analyzed.

The sequences from maize show sequence similarity to the published barley Mlo sequence as provided in Tables 1 and 2. FIG. 1 provides a sequence alignment for the maize Mlo homologues with the barley Mlo sequence designated as HvMlo1. FIG. 2 groups the Mlo sequences based on relatedness as well as the plant tissue from which it was isolated.

The ZmMLO genes were mapped by RFLP analysis using Southern blots of genomic DNA isolated from F2 and F3 and F4 segregating maize populations. The DNA was isolated using a modified CTAB adapted from the CERES RFLP Lab Manuel based on the Saghai-Mahoof procedure (Saghai-Mahoof et al. (1984) Proc. Natl. Acad. Sci. USA 81:8014-8018). Genomic DNA was cut with four restriction enzymes BamHI, EcoRI, EcoRV, and HindIII and run (5 μg/lane) on 0.8% TAE agarose gels for 800 volt-hours. The gels were Southern blot transferred to Hybond-N membrane (Amersham Life Science), baked and UV-crosslinked. The ZmMLO cDNA inserts were liberated from its vector by cleaving with two restriction enzymes, usually SalI and NotI, and the insert was purified from agarose gels following electrophoresis. The clone was random prime labeled with 32 P-dCTP using the RTS (Gibco BRL) labeling kit. The cDNA inserts were used to probe parental screening blots to identify a mapping population with a RFLP polymorphism. Once a polymorphism was identified, the inserts were used to probe mapping blots containing DNA from segregating individuals. The map position was determined using MAPMAKER/EXP 3.0 (Lander et al (1987) Genomics 1:174-181) by scoring 86 segregating progeny as homozygous parent A, as homozygous parent B, or as heterozygous. The map position was assigned to an existing core RFLP map of either of the following three populations: ALEB9 (240 individuals) DRAG2 (283 individuals) or MARS (1075 individuals). Table 3 shows the association of maize Mlo homologues and disease related loci and QTLS.

The Trait Utility System for corn (TUSC) system was utilized to generate transposon mutants in the Mlo sequences in maize. The combination of transposon mutagenesis via Mutator and PCR-based selection of target-specific Mu insertions makes up the TUSC system. See, for example, Benson et al. (1995) Plant Cell . 7:75-84; Mena et al. (1996) Science 274:1537-1540; U.S. patent application Ser. No. 08/835,638 and U.S. patent application Ser. No. 08/262,056; all of which are herein incorporated by reference. Table 4 shows Mutator insertion mutants of ZmMlo homologues.

Oligonucleotide primers based on Mlo maize sequences were designed and paired with Mu TIR primer in PCR reactions. The products of the screening process identify individuals in the collection having Mu elements inserted into Mlo sequences.

The gene-specific primers used for TUSC of ZmMLO2 are: 19005, 5′-AGTTATCCAAGGCATGCCGGTGGTAAAACT-3′ (SEQ ID NO18); and 19007, 5′-AAGTCATCGAGGGAGGCATCACTTACAG-3′ (SEQ ID NO: 19 ). For TUSC the mutator-element specific primer used was: 9242, 5′-AGAGAAGCCAACGCCA(AT)CGCCTC(CT)ATTTCGTC-3′ (SEQ ID NO:20). Seven positive “hits” to ZmMLO2 were identified. Twenty F2 seeds for each of the hits were planted in the greenhouse and screened as follows. Seeds were planted in trays, containing 2-inch wells for each seed, in Strong-Lite (circleR) Universal Mix potting soil (Universal Mix, Pine Bluff, Ark., U.S.A.) and grown in a greenhouse (16 hour day, 20.6-34.8° C., 50% average RH, 560-620 microEinsteins, of light both sun and halogen lamps, averaging 25 heat units per day. Plants were grown for 14-18 days until the V5 stage (ca. 25 cm height) and inoculated with spores of Cochliobolus heterostrophus (Drechs.) Dreshs. Race 0, also known as Bipolaris maydis (Southern Leaf Blight) and Cochliobolus carbonum R. R Nelson, also known as Bipolaris zeicola (Helminthosporium Leaf Spot). For general leaf inoculation, spore suspensions of 4×10 4 per ml of 0.02% Tween-20 were sprayed as an aerosol on the leaves. Approximately 0.5 ml were applied per leaf late in the afternoon. The plants were then immediately covered with a plastic tent and kept at R.T. in order to enhance humidity and spore germination. The plastic tent was removed early in the morning, and the plants were returned to the greenhouse for the duration of the experiment, usually three days. Susceptibility to Cochliobolus carbonum was determined by either increased size or number of lesions. Resistance to Cochliobolus heterostrophus was determined by either reduced size or number of lesions. Lesion mimics were also investigated; these mutations form spontaneous lesions in the absence of pathogen attach. Other phenotypes affecting growth and development were also noted.

Transformation and Regeneration of Transgenic Plants

Immature maize embryos from greenhouse donor plants are bombarded with a plasmid containing the ZmMLO1 antisense sequence operably linked to the ubiquitin promoter (FIG. 3 ). This plasmid also contains the selectable marker gene PAT (Wohlleben et al. (1988) Gene 70:25-37) that confers resistance to the herbicide Bialophos. Transformation is performed as follows. All media recipes are in the Appendix.

Preparation of Target Tissue

The ears are surface sterilized in 30% Chlorox bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two times with sterile water. The immature embryos are excised and placed embryo axis side down (scutellum side up), 25 embryos per plate, on 560Y medium for 4 hours and then aligned within the 2.5-cm target zone in preparation for bombardment.

Preparation of DNA

A plasmid vector comprising the PAT selectable marker and the ZmMlo1 antisense sequence operably linked to a the ubiquitin is made and precipitated onto 1.1 μm (average diameter) tungsten pellets using a CaCl 2 precipitation procedure as follows:

100 μl prepared tungsten particles in water

10 μl (1 μg) DNA in TrisEDTA buffer (1 μg total)

100 μl 2.5 M CaCl 2

10 μl 0.1 M spermidine

Each reagent is added sequentially to the tungsten particle suspension, while maintained on the multitube vortexer. The final mixture is sonicated briefly and allowed to incubate under constant vortexing for 10 minutes. After the precipitation period, the tubes are centrifuged briefly, liquid removed, washed with 500 ml 100% ethanol, and centrifuged for 30 seconds. Again the liquid is removed, and 105 μl 100% ethanol is added to the final tungsten particle pellet. For particle gun bombardment, the tungsten/DNA particles are briefly sonicated and 10 μl spotted onto the center of each macrocarrier and allowed to dry about 2 minutes before bombardment.

Particle Gun Treatment

The sample plates are bombarded at level #4 in particle gun #HE34- 1 or #HE34-2. All samples receive a single shot at 650 PSI, with a total of ten aliquots taken from each tube of prepared particles/DNA.

Subsequent Treatment

Following bombardment, the embryos are kept on 560Y medium for 2 days, then transferred to 560R selection medium containing 3 mg/liter Bialaphos, and subcultured every 2 weeks. After approximately 10 weeks of selection, selection-resistant callus clones are transferred to 288J medium to initiate plant regeneration. Following somatic embryo maturation (2-4 weeks), well-developed somatic embryos are transferred to medium for germination and transferred to the lighted culture room. Approximately 7-10 days later, developing plantlets are transferred to 272V hormone-free medium in tubes for 7-10 days until plantlets are well established. Plants are then transferred to inserts in flats (equivalent to 2.5″ pot) containing potting soil and grown for 1 week in a growth chamber, subsequently grown an additional 1-2 weeks in the greenhouse, then transferred to classic 600 pots (1.6 gallon) and grown to maturity. Plants are monitored and scored for enhanced disease resistance as a result of antisense disruption of ZmMlo1function.

Directions:

@=Add after bringing up to volume

Dissolve ingredients in polished D-I H 2 O in sequence

Adjust to pH 5.6

Bring up to volume with polished D-I H 2 O after adjusting pH

Sterilize and cool to 60° C.

##=Dissolve 0.100 g of Nicotinic Acid; 0.020 g of Thiamine.HCL; 0.100 g of Pyridoxine.HCL; and 0.400 g of Glycine in 875.00 ml of polished D-I H 2 O in sequence. Bring up to volume with polished D-I H 2 O. Make in 400 ml portions. Thiamine.HCL & Pyridoxine.HCL are in Dark Desiccator. Store for one month, unless contamination or precipitation occurs, then make fresh stock.

Total Volume (L)=1.00

Directions:

@=Add after bringing up to volume

Dissolve ingredients in polished D-I H 2 O in sequence

Adjust to pH 5.6

Bring up to volume with polished D-I H 2 O after adjusting pH

Sterilize and cool to 60° C.

Add 3.5g/L of Gelrite for cell biology.

##=Dissolve 0.100 g of Nicotinic Acid; 0.020 g of Thiamine.HCL; 0.100 g of

Pyridoxine.HCL; and 0.400 g of Glycine in 875.00 ml of polished D-I H 2 O in sequence.

Bring up to volume with polished D-I H 2 O. Make in 400 ml portions. Thiamine.HCL & Pyridoxine.HCL are in Dark Desiccator. Store for one month, unless contamination or precipitation occurs, then make fresh stock.

Total Volume (L)=1.00

Directions:

@=Add after bringing up to volume

#=Add after sterilizing and cooling to temp.

Dissolve ingredients in D-I H 2 O in sequence

Adjust to pH 5.8 with KOH

Bring up to volume with D-I H 2 O

Sterilize and cool to room temp.

Total Volume (L)=1.00

Directions:

@=Add after bringing up to volume

#=Add after sterilizing and cooling to temp.

Dissolve ingredients in D-I H 2 O in sequence

Adjust to pH 5.8 with KOH

Bring up to volume with D-I H 2 O

Sterilize and cool to room temp.

** Autoclave less time because of increased sucrose**

Total Volume (L)=1.00

All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

20

1

845

Zea mays

Mlo1

1

aggagataca ggacaaggcg acggtcatca agggggcgcc tgtggtggag ccaagtgaca 60

ggttcttctg gtttaaccgc cctggctggg tcctcttcct catccacctc acgctcttcc 120

agaacgcctt ccagatggcg catttcgttt ggacactgct caccccagac ctgaagaaat 180

gctaccacga gaggctgggc ctgagcatca tgaaagttgc ggtggggctg gttctccagg 240

tcctctgcag ctacatcacc ttcccgctct acgcgctcgt cacgcagatg gggtcgcaca 300

tgaagaagac catcttcgag gagcagacgg ccaaggcggt gatgaagtgg cgcaagacgg 360

ccaaggataa ggtgcggcag cgggaggcgg caggcttcct cgacgtgctg acgagcgccg 420

acaccacgcc gagccacagc cgcgcgacgt cgccgagccg gggcaactcg ccggtgcacc 480

tgctccacaa gtacaggggc aggtcggagg aaccgcagag cgggccggcg tcgccggggc 540

gggagctcgg ggacatgtac ccggtggctg accagcatcg cctgcacagg ctggaccccg 600

agaggatgag gcccgcctcg tccaccgccg tcaacattga catcgctgat gccgattttt 660

cttttagcat gcggtgacct gaccttgaac gaattctgtg tccttactct tgtataggga 720

agcaaaagca tagacggaga acataatgac acgttacgtt agggaaagtt tcgtttattc 780

atcataaaat agaatacgta ataaactagt atctccctct tcaaaaaaaa aaaaaaaaaa 840

aaaaa 845

2

224

Zea mays

Mlo1

2

Glu Ile Gln Asp Lys Ala Thr Val Ile Lys Gly Ala Pro Val Val Glu

1 5 10 15

Pro Ser Asp Arg Phe Phe Trp Phe Asn Arg Pro Gly Trp Val Leu Phe

20 25 30

Leu Ile His Leu Thr Leu Phe Gln Asn Ala Phe Gln Met Ala His Phe

35 40 45

Val Trp Thr Leu Leu Thr Pro Asp Leu Lys Lys Cys Tyr His Glu Arg

50 55 60

Leu Gly Leu Ser Ile Met Lys Val Ala Val Gly Leu Val Leu Gln Val

65 70 75 80

Leu Cys Ser Tyr Ile Thr Phe Pro Leu Tyr Ala Leu Val Thr Gln Met

85 90 95

Gly Ser His Met Lys Lys Thr Ile Phe Glu Glu Gln Thr Ala Lys Ala

100 105 110

Val Met Lys Trp Arg Lys Thr Ala Lys Asp Lys Val Arg Gln Arg Glu

115 120 125

Ala Ala Gly Phe Leu Asp Val Leu Thr Ser Ala Asp Thr Thr Pro Ser

130 135 140

His Ser Arg Ala Thr Ser Pro Ser Arg Gly Asn Ser Pro Val His Leu

145 150 155 160

Leu His Lys Tyr Arg Gly Arg Ser Glu Glu Pro Gln Ser Gly Pro Ala

165 170 175

Ser Pro Gly Arg Glu Leu Gly Asp Met Tyr Pro Val Ala Asp Gln His

180 185 190

Arg Leu His Arg Leu Asp Pro Glu Arg Met Arg Pro Ala Ser Ser Thr

195 200 205

Ala Val Asn Ile Asp Ile Ala Asp Ala Asp Phe Ser Phe Ser Met Arg

210 215 220

3

2030

Zea mays

Mlo2

3

gcggcaagca attgtctcat ctgctccact ccatctccat ttaacgcact ctccactcct 60

tcgcggcggg ggtttaggcg cacggcggca ggccaattaa cgccgctgcg gcgagcaccg 120

ggggggaaat gggcggcgac ggcacgaggg cgctggacca gacgccgacg tgggccgtgg 180

ccgccgtctg cgctgtcatc gtcgcggcgt ccatactcct cgagggcttc ctccaccacc 240

tcggtcagtt gctcaacaag aagcggaaga aggcgctgtt tgacgctctg gagaaggtta 300

aatcggagct gatgactctg gggttcatat cgctgctgct gaccgtgacg ggaaggtaca 360

tcgcgcgcat ctgcatcccg gagggagccg caaacaccat gctgccctgc cgcctgtccg 420

gtcactcggt ggcggaagag cccaagggcc atggccgacg gcacctatct gaagatccaa 480

ccaacctttt ttcctgccgc aaaggcatgg tgtcgcttgt ttcagccgac ggcatgcatc 540

agctgcacat tttcgtgttc ttcttggccg tcttccatgt taccttcagt ttcttcacaa 600

tgtccttggg tagagcaaag actcgtatat ggaaggtgtg ggaaaaggaa acttgctccc 660

ctcagtataa ttatttaaat gacccctcaa agttcaggct tacgcaccaa acatcttttg 720

tgaggcaaca tgcaagttgt tggagcaaaa gcacaatcac gctctatttt gtgagcttct 780

ttaggcagtt cttcagatcc gtccgtaaga cagactactt tactttgcga catggattca 840

tttcagctca tttatctccg gggactaggt tcaattttcg aaagtacatt aaaaggtcct 900

tggaggatga tttcaagaca gttgttggca ttagtccacc actatgggct tctgctttgg 960

ctgtcatgct gttcaatgtt catggatggc acaacttatt ctggttctct gcaattcccc 1020

ttgtagttat tctagcagtt ggaacaaagc tgcaggctat aattgctatg atggctattg 1080

aaattgcaga gaggcataca gttatccaag gcatgccggt ggtaaaacta agtgatgatc 1140

atttctggtt cgggaagcct cgtttggttc tccacctcat tcatttcgca tcatttcaga 1200

acgcatttga aattacatat ttcttttgga tctggtacga atttggcctg aggtcctgct 1260

tccatgacaa ctttgagttt atcattgcaa gagtctgcct tggggctatc gtccagttta 1320

tgtgcagcta catcaccctt ccactctatg cccttgtttc tcagatgggt tcagaaatga 1380

agcgaacaat tttcgacgag cagacagcga aggccctgaa gaaatggcac aaggccgtgg 1440

tgaagaagaa acaccacaag gattcctcac acaattcttc cgagactcct agcactgaca 1500

caacaggacc tgcaggggaa gcgggtgagt ggcagcggct gcacgaggtg ccggtccggc 1560

acctccaccg gtacaagacc atcgcgcacg tcggcggcgt gaggagcccg ttgtcggacc 1620

cggactacag cgacacggac gacaccgagc cgctgtcgtt gcagactagg cacctgatac 1680

cgccggcgaa gcagcggagc ctcgacaccg agcgcgcgga ggtgcgcgtg aacgtcgtag 1740

agacagcggc ggcgccaagc gacgtcctcc aagacagctt ctcgttccca aggctgcttc 1800

ctcctcgcca tgtgccggac aagtaaagtg gccagcagtc tgcttagtac tacagactgt 1860

cctatataga gatagtagaa tagtagtttc aagttataag tcgatttggc tagttataca 1920

acttttgtta tttatttaga taaaggctat gtctatatag caaaagttgt aaaactagaa 1980

aaatcaaaat gatttataat ttgaaaaaaa aaaaaaaaaa aaaaaaaaaa 2030

4

565

Zea mays

Mlo2

4

Met Gly Gly Asp Gly Thr Arg Ala Leu Asp Gln Thr Pro Thr Trp Ala

1 5 10 15

Val Ala Ala Val Cys Ala Val Ile Val Ala Ala Ser Ile Leu Leu Glu

20 25 30

Gly Phe Leu His His Leu Gly Gln Leu Leu Asn Lys Lys Arg Lys Lys

35 40 45

Ala Leu Phe Asp Ala Leu Glu Lys Val Lys Ser Glu Leu Met Thr Leu

50 55 60

Gly Phe Ile Ser Leu Leu Leu Thr Val Thr Gly Arg Tyr Ile Ala Arg

65 70 75 80

Ile Cys Ile Pro Glu Gly Ala Ala Asn Thr Met Leu Pro Cys Arg Leu

85 90 95

Ser Gly His Ser Val Ala Glu Glu Pro Lys Gly His Gly Arg Arg His

100 105 110

Leu Ser Glu Asp Pro Thr Asn Leu Phe Ser Cys Arg Lys Gly Met Val

115 120 125

Ser Leu Val Ser Ala Asp Gly Met His Gln Leu His Ile Phe Val Phe

130 135 140

Phe Leu Ala Val Phe His Val Thr Phe Ser Phe Phe Thr Met Ser Leu

145 150 155 160

Gly Arg Ala Lys Thr Arg Ile Trp Lys Val Trp Glu Lys Glu Thr Cys

165 170 175

Ser Pro Gln Tyr Asn Tyr Leu Asn Asp Pro Ser Lys Phe Arg Leu Thr

180 185 190

His Gln Thr Ser Phe Val Arg Gln His Ala Ser Cys Trp Ser Lys Ser

195 200 205

Thr Ile Thr Leu Tyr Phe Val Ser Phe Phe Arg Gln Phe Phe Arg Ser

210 215 220

Val Arg Lys Thr Asp Tyr Phe Thr Leu Arg His Gly Phe Ile Ser Ala

225 230 235 240

His Leu Ser Pro Gly Thr Arg Phe Asn Phe Arg Lys Tyr Ile Lys Arg

245 250 255

Ser Leu Glu Asp Asp Phe Lys Thr Val Val Gly Ile Ser Pro Pro Leu

260 265 270

Trp Ala Ser Ala Leu Ala Val Met Leu Phe Asn Val His Gly Trp His

275 280 285

Asn Leu Phe Trp Phe Ser Ala Ile Pro Leu Val Val Ile Leu Ala Val

290 295 300

Gly Thr Lys Leu Gln Ala Ile Ile Ala Met Met Ala Ile Glu Ile Ala

305 310 315 320

Glu Arg His Thr Val Ile Gln Gly Met Pro Val Val Lys Leu Ser Asp

325 330 335

Asp His Phe Trp Phe Gly Lys Pro Arg Leu Val Leu His Leu Ile His

340 345 350

Phe Ala Ser Phe Gln Asn Ala Phe Glu Ile Thr Tyr Phe Phe Trp Ile

355 360 365

Trp Tyr Glu Phe Gly Leu Arg Ser Cys Phe His Asp Asn Phe Glu Phe

370 375 380

Ile Ile Ala Arg Val Cys Leu Gly Ala Ile Val Gln Phe Met Cys Ser

385 390 395 400

Tyr Ile Thr Leu Pro Leu Tyr Ala Leu Val Ser Gln Met Gly Ser Glu

405 410 415

Met Lys Arg Thr Ile Phe Asp Glu Gln Thr Ala Lys Ala Leu Lys Lys

420 425 430

Trp His Lys Ala Val Val Lys Lys Lys His His Lys Asp Ser Ser His

435 440 445

Asn Ser Ser Glu Thr Pro Ser Thr Asp Thr Thr Gly Pro Ala Gly Glu

450 455 460

Ala Gly Glu Trp Gln Arg Leu His Glu Val Pro Val Arg His Leu His

465 470 475 480

Arg Tyr Lys Thr Ile Ala His Val Gly Gly Val Arg Ser Pro Leu Ser

485 490 495

Asp Pro Asp Tyr Ser Asp Thr Asp Asp Thr Glu Pro Leu Ser Leu Gln

500 505 510

Thr Arg His Leu Ile Pro Pro Ala Lys Gln Arg Ser Leu Asp Thr Glu

515 520 525

Arg Ala Glu Val Arg Val Asn Val Val Glu Thr Ala Ala Ala Pro Ser

530 535 540

Asp Val Leu Gln Asp Ser Phe Ser Phe Pro Arg Leu Leu Pro Pro Arg

545 550 555 560

His Val Pro Asp Lys

565

5

1841

Zea mays

Mlo4

5

agatatcagg gcagctggaa ctgaaggtgg cgggagccga gtgatccggc ggtgagctga 60

gcggggcggg atggcggcgg agcaggggcg gtcgctggcg gagacgccca cctggtccgt 120

ggcaaccgtc accacgctca tggtcgctgc ctgcttcctc gtcgagcgct ccctctcgcg 180

cttcgccaag tggctgcgca agaccaagcg gaaggccatg ctcgccgcgc tcgagaagat 240

ccgcgaagag ctgatgctgc tcggagtcat ctcgctgctg ctcagccaga cggcgcgctt 300

catatcggag atctgcgtgc cgtcctcgct cttcaccagc cgcttctaca tctgctccga 360

gagcgactac caggacctgc tgcgcaacac ggacgccaac cagacggcgc tcgacaagaa 420

catgttcggt ggccaacggc tgcacgtctg tggcgagggc catgaacctt ttgtttcgta 480

cgagggcctt gagcagctgc accggtttct cttcatcctt ggtatcactc atgtgttgta 540

cagttttgta acagtggttc tgtccatgat caagatctat agctggagga agtgggaaac 600

cttagcaggt ccaattgctg ctgaggaatt gaaagctagg agaaccaagg tgatgagaag 660

gcagtcaacc tttgttttta acaatgcttc tcatccatgg agcaaaaata aaatacttat 720

ttggatgctt tgctttttgc gtcaattcaa gggctccata ataaggtcag actatttggc 780

actgaggttg ggctttgtca catatcacaa gctaccacat tcatatgact tccataaata 840

catggtacgg agcatggaag atgattacaa tgggactatt ggtatcagtt ggccactttg 900

ggcatatgcg attgtctgca tattaatcaa tgttcatggt atcaatatat atttctggtt 960

gtcctttgtt cctgttattc tggtgcttct agtgggtact gaacttcagc acgtcattgc 1020

tcagttggct ttggaagtcg ctgaggcaac agcgccttat gttggctcac aacttaaact 1080

gcgtgatgat ctattttggt ttggaaagcc tcgggtactc tggtggctta tacagttcat 1140

ttcatttcag aatgcttttg agctggcaac attcttatgg tctctgtggg aactcagtgc 1200

acaaacatgt ttcatgaagc actactacat ggttgccatt cggttgattt ctgggctcct 1260

agttcagttt tggtgcagct acagcacact gccgctgaat gtgattattt ctcagatggg 1320

ttccaagttc aagaaatcac tggtctcgga gaacgtgagg gagtcgctgc acagctggtg 1380

caagagggtt aaggacagga gccgacacaa tccgctcttc tcgcggaacg ggaccctcac 1440

gaccagatcc gtgtgctccc tagacaccac ctacgagacg gatcacgaga cgaacacggt 1500

gtgcacgctg tcgaggacgg cgtcggcgac gtcgctggac gaccagttga ccgtggtcac 1560

cgtcgatgac gagccgtcct gcattgagaa ggatgtctga cgcagttgct gattcgccaa 1620

aactacatac tgcaccgacc tgtgtgttag gaggtactct aggactgaaa tgaatgtata 1680

tgtgcagtgg ttgcccctat cgtggcagtg accatgtgta tatatagaac taaaaaaact 1740

ataatgtctc ttagctgaga gtgagaggct gtacatggaa atacttgagg cagcttggac 1800

caaatacaaa actgcatctt ttcgagaaaa aaaaaaaaaa a 1841

6

509

Zea mays

Mlo4

6

Met Ala Ala Glu Gln Gly Arg Ser Leu Ala Glu Thr Pro Thr Trp Ser

1 5 10 15

Val Ala Thr Val Thr Thr Leu Met Val Ala Ala Cys Phe Leu Val Glu

20 25 30

Arg Ser Leu Ser Arg Phe Ala Lys Trp Leu Arg Lys Thr Lys Arg Lys

35 40 45

Ala Met Leu Ala Ala Leu Glu Lys Ile Arg Glu Glu Leu Met Leu Leu

50 55 60

Gly Val Ile Ser Leu Leu Leu Ser Gln Thr Ala Arg Phe Ile Ser Glu

65 70 75 80

Ile Cys Val Pro Ser Ser Leu Phe Thr Ser Arg Phe Tyr Ile Cys Ser

85 90 95

Glu Ser Asp Tyr Gln Asp Leu Leu Arg Asn Thr Asp Ala Asn Gln Thr

100 105 110

Ala Leu Asp Lys Asn Met Phe Gly Gly Gln Arg Leu His Val Cys Gly

115 120 125

Glu Gly His Glu Pro Phe Val Ser Tyr Glu Gly Leu Glu Gln Leu His

130 135 140

Arg Phe Leu Phe Ile Leu Gly Ile Thr His Val Leu Tyr Ser Phe Val

145 150 155 160

Thr Val Val Leu Ser Met Ile Lys Ile Tyr Ser Trp Arg Lys Trp Glu

165 170 175

Thr Leu Ala Gly Pro Ile Ala Ala Glu Glu Leu Lys Ala Arg Arg Thr

180 185 190

Lys Val Met Arg Arg Gln Ser Thr Phe Val Phe Asn Asn Ala Ser His

195 200 205

Pro Trp Ser Lys Asn Lys Ile Leu Ile Trp Met Leu Cys Phe Leu Arg

210 215 220

Gln Phe Lys Gly Ser Ile Ile Arg Ser Asp Tyr Leu Ala Leu Arg Leu

225 230 235 240

Gly Phe Val Thr Tyr His Lys Leu Pro His Ser Tyr Asp Phe His Lys

245 250 255

Tyr Met Val Arg Ser Met Glu Asp Asp Tyr Asn Gly Thr Ile Gly Ile

260 265 270

Ser Trp Pro Leu Trp Ala Tyr Ala Ile Val Cys Ile Leu Ile Asn Val

275 280 285

His Gly Ile Asn Ile Tyr Phe Trp Leu Ser Phe Val Pro Val Ile Leu

290 295 300

Val Leu Leu Val Gly Thr Glu Leu Gln His Val Ile Ala Gln Leu Ala

305 310 315 320

Leu Glu Val Ala Glu Ala Thr Ala Pro Tyr Val Gly Ser Gln Leu Lys

325 330 335

Leu Arg Asp Asp Leu Phe Trp Phe Gly Lys Pro Arg Val Leu Trp Trp

340 345 350

Leu Ile Gln Phe Ile Ser Phe Gln Asn Ala Phe Glu Leu Ala Thr Phe

355 360 365

Leu Trp Ser Leu Trp Glu Leu Ser Ala Gln Thr Cys Phe Met Lys His

370 375 380

Tyr Tyr Met Val Ala Ile Arg Leu Ile Ser Gly Leu Leu Val Gln Phe

385 390 395 400

Trp Cys Leu Tyr Ser Thr Leu Pro Leu Asn Val Ile Ile Ser Gln Met

405 410 415

Gly Ser Lys Phe Lys Lys Ser Leu Val Ser Glu Asn Val Arg Glu Ser

420 425 430

Leu His Ser Trp Cys Lys Arg Val Lys Asp Arg Ser Arg His Asn Pro

435 440 445

Leu Phe Ser Arg Asn Gly Thr Leu Thr Thr Arg Ser Val Cys Ser Leu

450 455 460

Asp Thr Thr Tyr Glu Thr Asp His Glu Thr Asn Thr Val Cys Thr Leu

465 470 475 480

Ser Arg Thr Ala Ser Ala Thr Ser Leu Asp Asp Gln Leu Thr Val Val

485 490 495

Thr Val Asp Asp Glu Pro Ser Cys Ile Glu Lys Asp Val

500 505

7

673

Zea mays

Mlo5

7

tgatccactt catcctcttc cagaacgcgt ttgagatcgc attcttcttc tggattctga 60

ctacctacgg tttcaactcc tgcatcatgg accacgtccc cttcattgtg ccgaggctcg 120

ttgtcggggc catcatccag ctcctctgta gctacagcac cctgcctctg tacgcgattg 180

tcacgcagat ggggactttc ttcaagaagg agatcttcga tgagcacgtg cagcagagcc 240

ttctgggctg ggcgcagaag gccaagaaga ggaaagcgct caggaacaat ggcaatggca 300

gcaatggcgc cgcggccggg tcgtcacatc catctgcgac tgcgaggctc gaactgatga 360

gacgagctgt cgctcttgaa gaaggcagtg ccggtggcaa cgggagtgag gctagtgcgg 420

ccgagctaca cgacacgggc ccgaagctct gaacgctgta gaaaacgccc ggcagagcgt 480

cagaagatgc agtgtttctg tacgcgagcc atatgtcact atgtcgtgtg gctgtttagc 540

caggtggtgt agtgtgatac tgcgtatcat gatttgtatg cttttctgga ttgagcttga 600

agcgtgttat ggagtgggga gctttgtgta gtgttgtgct caaaaaaaaa aaaaaaaaaa 660

aaaaaaaaaa aaa 673

8

149

Zea mays

Mlo5

8

Ile His Phe Ile Leu Phe Gln Asn Ala Phe Glu Ile Ala Phe Phe Phe

1 5 10 15

Trp Ile Leu Thr Thr Tyr Gly Phe Asn Ser Cys Ile Met Asp His Val

20 25 30

Pro Phe Ile Val Pro Arg Leu Val Val Gly Ala Ile Ile Gln Leu Leu

35 40 45

Cys Ser Tyr Ser Thr Leu Pro Leu Tyr Ala Ile Val Thr Gln Met Gly

50 55 60

Thr Phe Phe Lys Lys Glu Ile Phe Asp Glu His Val Gln Gln Ser Leu

65 70 75 80

Leu Gly Trp Ala Gln Lys Ala Lys Lys Arg Lys Ala Leu Arg Asn Asn

85 90 95

Gly Asn Gly Ser Asn Gly Ala Ala Ala Gly Ser Ser His Pro Ser Ala

100 105 110

Thr Ala Arg Leu Glu Leu Met Arg Arg Ala Val Ala Leu Glu Glu Gly

115 120 125

Ser Ala Gly Gly Asn Gly Ser Glu Ala Ser Ala Ala Glu Leu His Asp

130 135 140

Thr Gly Pro Lys Leu

145

9

1815

Zea mays

Mlo6

9

aagcgatcga tagaataaat taaaggcgcg ggggcaacaa caagaatggg gggcggtggc 60

ggtggcggca actcgcggga gcttgaccag acgccgacat gggcggtggc gtcggtgtgc 120

ggcgtgatcg tgctcatctc catcctgctg gagaaggggc tccaccacgt gggcgagttc 180

ttctcccacc gcaagaagaa ggccatggtg gaggccctgg agaaggtgaa ggcggagctc 240

atggtgctgg gcttcatctc gctcctcctc gtgttcggcc agaactacat catcaaggtc 300

tgcatcagca accacgccgc caacaccatg ctcccctgca agctcgaggc cgccgccgtc 360

gagggcaagg acggccacgg caaggaggcc gccgccgtgg tcgctggcaa gaagaaggtc 420

gccgtcgccg tccctggaaa gaagaagaag aaggccgccg ccgccgccga ccatcttggc 480

ggtgtggtgg actggccgcc gccctactac gcgcacaacg ccaggatgct ggcggaggcg 540

agcatggcga ccaagtgccc cgaggggaaa gtgccgctca tctccatcaa cgccctgcac 600

cagctgcaca tcttcatctt cttcctcgcc gtcttccacg tctcctacag cgcaatcacc 660

atggcgctcg gcagggccaa gatacgtgca tggaaagagt gggagaaaga agctgcagga 720

caagactacg agttctcaca tgacccgacg cggttcaggt tcacccacga gacttccttc 780

gtgaggcagc atatgaatgt gctgaacaag ttcccagcat cattctacat cagcaacttc 840

ttccggcagt tcttcaggtc cgtgaggcag gcagactact gcgcgctgcg ccacagcttt 900

gtcaacgtcc atctggcccc tggcagcaag tttgatttcc agaagtacat caagcggtct 960

ctggaggatg acttcaaggt gatcgtgggg atcagtcctc ctctgtgggc ttctgctctc 1020

atcttcctct tcctcaacgt caatggatgg cacaccatgc tctggatctc catcatgccg 1080

gtggtgatca tcctgtcggt ggggacgaag ctgcagggca tcatctgccg catggcgatc 1140

gacatcacgg agcgccacgc cgtcatccag ggcatcccga tggtgcaagt cagcgactcc 1200

tacttctggt tcgcacgccc caccttcgtg ctcttcctca tccacttcac cctcttccag 1260

aatggcttcc agatcatcta cttcctctgg attctgtatg agtacggcat ggactcgtgc 1320

ttcaacgact ccgaagagtt cgtctttgca cgactctgcc ttggcgtggt tgtccaggtg 1380

ctgtgcagct acgtgacgct cccgctgtac gcgctcgtct cccaaatggg ctccaccatg 1440

aagcagtcca tcttcgacga gcagacctcc aaggcgctca agaactggcg cgccggcgcc 1500

aagaagaagg ctcccaccgg cggctccaag cacggcggtg gtggctcccc caccgccggc 1560

ggcagcccca ccaaggccga cggcgacgcg tagagaggaa cacgctaact ttaatttctg 1620

tgtgcttaat tgcctaggct cgttaagtca gaacatgcat gcatgtaaca ccactgctgg 1680

ttttcatata gtgtcgacag atggtcaacg tactttttgc gatcccactt gtattttttt 1740

ttacaatgaa gcacccgtcc gcgtccgtgg acactgcaag tgcattgcca aaaaaaaaaa 1800

aaaaaaaaaa aaaaa 1815

10

515

Zea mays

Mlo6

10

Met Gly Gly Gly Gly Gly Gly Gly Asn Ser Arg Glu Leu Asp Gln Thr

1 5 10 15

Pro Thr Trp Ala Val Ala Ser Val Cys Gly Val Ile Val Leu Ile Ser

20 25 30

Ile Leu Leu Glu Lys Gly Leu His His Val Gly Glu Phe Phe Ser His

35 40 45

Arg Lys Lys Lys Ala Met Val Glu Ala Leu Glu Lys Val Lys Ala Glu

50 55 60

Leu Met Val Leu Gly Phe Ile Ser Leu Leu Leu Val Phe Gly Gln Asn

65 70 75 80

Tyr Ile Ile Lys Val Cys Ile Ser Asn His Ala Ala Asn Thr Met Leu

85 90 95

Pro Cys Lys Leu Glu Ala Ala Ala Val Glu Gly Lys Asp Gly His Gly

100 105 110

Lys Glu Ala Ala Ala Val Val Ala Gly Lys Lys Lys Val Ala Val Ala

115 120 125

Val Pro Gly Lys Lys Lys Lys Lys Ala Ala Ala Ala Ala Asp His Leu

130 135 140

Gly Gly Val Val Asp Trp Pro Pro Pro Tyr Tyr Ala His Asn Ala Arg

145 150 155 160

Met Leu Ala Glu Ala Ser Met Ala Thr Lys Cys Pro Glu Gly Lys Val

165 170 175

Pro Leu Ile Ser Ile Asn Ala Leu His Gln Leu His Ile Phe Ile Phe

180 185 190

Phe Leu Ala Val Phe His Val Ser Tyr Ser Ala Ile Thr Met Ala Leu

195 200 205

Gly Arg Ala Lys Ile Arg Ala Trp Lys Glu Trp Glu Lys Glu Ala Ala

210 215 220

Gly Gln Asp Tyr Glu Phe Ser His Asp Pro Thr Arg Phe Arg Phe Thr

225 230 235 240

His Glu Thr Ser Phe Val Arg Gln His Met Asn Val Leu Asn Lys Phe

245 250 255

Pro Ala Ser Phe Tyr Ile Ser Asn Phe Phe Arg Gln Phe Phe Arg Ser

260 265 270

Val Arg Gln Ala Asp Tyr Cys Ala Leu Arg His Ser Phe Val Asn Val

275 280 285

His Leu Ala Pro Gly Ser Lys Phe Asp Phe Gln Lys Tyr Ile Lys Arg

290 295 300

Ser Leu Glu Asp Asp Phe Lys Val Ile Val Gly Ile Ser Pro Pro Leu

305 310 315 320

Trp Ala Ser Ala Leu Ile Phe Leu Phe Leu Asn Val Asn Gly Trp His

325 330 335

Thr Met Leu Trp Ile Ser Ile Met Pro Val Val Ile Ile Leu Ser Val

340 345 350

Gly Thr Lys Leu Gln Gly Ile Ile Cys Arg Met Ala Ile Asp Ile Thr

355 360 365

Glu Arg His Ala Val Ile Gln Gly Ile Pro Met Val Gln Val Ser Asp

370 375 380

Ser Tyr Phe Trp Phe Ala Arg Pro Thr Phe Val Leu Phe Leu Ile His

385 390 395 400

Phe Thr Leu Phe Gln Asn Gly Phe Gln Ile Ile Tyr Phe Leu Trp Ile

405 410 415

Leu Tyr Glu Tyr Gly Met Asp Ser Cys Phe Asn Asp Ser Glu Glu Phe

420 425 430

Val Phe Ala Arg Leu Cys Leu Gly Val Val Val Gln Val Leu Cys Ser

435 440 445

Tyr Val Thr Leu Pro Leu Tyr Ala Leu Val Ser Gln Met Gly Ser Thr

450 455 460

Met Lys Gln Ser Ile Phe Asp Glu Gln Thr Ser Lys Ala Leu Lys Asn

465 470 475 480

Trp Arg Ala Gly Ala Lys Lys Lys Ala Pro Thr Gly Gly Ser Lys His

485 490 495

Gly Gly Gly Gly Ser Pro Thr Ala Gly Gly Ser Pro Thr Lys Ala Asp

500 505 510

Gly Asp Ala

515

11

1660

Zea mays

Mlo7

11

gctagcagga gccaggtcgc gccgacagca gcagttggaa gaggatccat ggggaaggag 60

gcgacgcttg cgttcacgcc cacctgggtg gtggccatcg tatgcctcgt catcgtctct 120

atctccctcg ccgctgagcg ctcactccat tacctcggca agtatctgga gtgtaagaag 180

cagaaagcgc ttttttcagc tctacagaga ctaaaagaag agctaatgct tctcggattc 240

atttcattcg tcctgagtct ctcccaaggt tttattgtta gcatttgcat tccagaaact 300

tctactgact tcatgcttcc atgcaataga ggcaactcca gagttgcaga agaaggtgct 360

aaaatttgca acaaaaaggg tgatgttccc ttgctatcac tggaggcatt acatcagctg 420

cacattttca tatttgtact tggtttagtc catgttgtgt tctgcgctac aacaatatca 480

tttagtgggg ccaagatgag aaagtggaag cattgggaaa cagaaattca cagagaagta 540

catgagaagt tacagcaaga aaagaatgaa ggacaagggt catcattgag tattgttgtg 600

ctacatcggg agcaccagga tgaatttgtc cacaagcgga caaagggatt ttggatgaag 660

ctcgctgttg caagctggat aactgcattc ttgaagcaat ttcaagattc agtgagtaaa 720

tcagattatg aagcacttag atcagctttt gtcgtggaac actatcccga gaaaccagat 780

attgatttcc acaaatacat gactcgcgct gttgaatatg agtttaaaag agttgttggt 840

atcagctggt atctgtggct ttttgtaatc ttattcctgc tgctgaatat aaatggatgg 900

cacacatact tctggttggc tttcttgcct ctatttctgt tacttattgt tggtgccaaa 960

ctagagcaca ttatcactcg gttggctcaa gaggcagcga tatcattatc aaataataca 1020

gaggaagttc cgaaaataaa gccatgcaag gaccatttct ggtttcacaa gcctgagcta 1080

gtccttcatt tgatccattt catcctgttc cagaattcgt tcgagattag ttttttcttc 1140

tggattctgg tatcagaagg tttcggttcg tgtatgatgg aacggaagcc ttatgtcatt 1200

tccagacttg ttatcggggt gatcatcgaa gtcatctgca gctatatcac gctgccacta 1260

tacgccatcg tgacccatat gaccggacag atcaagctgc atggttttgg ctcccgcgtg 1320

cacgagagtg tccatggctg gatcggcttg aggaagaaac ccttctcgtt ctggaagatt 1380

cccggcggtg accctaacgc tgattccggc agggaggccg acgtaacccg cagggtggcc 1440

aaggagcggt cggggagctc gcgcagcatg ccgatggcgc cggctgacga gatcgtcacc 1500

gtcgacgacg tcgcggtggc tgcagctgcc gttggccaag gaccataaaa ttcctagcta 1560

gcaaacacca gcagggaagg caggcaggct gccgaggtac cgtgcgcagc gggttgatgg 1620

gtcggacgct gcggaaaatt cctcgtgtcg ggagcaggct 1660

12

499

Zea mays

Mlo7

12

Met Gly Lys Glu Ala Thr Leu Ala Phe Thr Pro Thr Trp Val Val Ala

1 5 10 15

Ile Val Cys Leu Val Ile Val Ser Ile Ser Leu Ala Ala Glu Arg Ser

20 25 30

Leu His Tyr Leu Gly Lys Tyr Leu Glu Cys Lys Lys Gln Lys Ala Leu

35 40 45

Phe Ser Ala Leu Gln Arg Leu Lys Glu Glu Leu Met Leu Leu Gly Phe

50 55 60

Ile Ser Phe Val Leu Ser Leu Ser Gln Gly Phe Ile Val Ser Ile Cys

65 70 75 80

Ile Pro Glu Thr Ser Thr Asp Phe Met Leu Pro Cys Asn Arg Gly Asn

85 90 95

Ser Arg Val Ala Glu Glu Gly Ala Lys Ile Cys Asn Lys Lys Gly Asp

100 105 110

Val Pro Leu Leu Ser Leu Glu Ala Leu His Gln Leu His Ile Phe Ile

115 120 125

Phe Val Leu Gly Leu Val His Val Val Phe Cys Ala Thr Thr Ile Ser

130 135 140

Phe Ser Gly Ala Lys Met Arg Lys Trp Lys His Trp Glu Thr Glu Ile

145 150 155 160

His Arg Glu Val His Glu Lys Leu Gln Gln Glu Lys Asn Glu Gly Gln

165 170 175

Gly Ser Ser Leu Ser Ile Val Val Leu His Arg Glu His Gln Asp Glu

180 185 190

Phe Val His Lys Arg Thr Lys Gly Phe Trp Met Lys Leu Ala Val Ala

195 200 205

Ser Trp Ile Thr Ala Phe Leu Lys Gln Phe Gln Asp Ser Val Ser Lys

210 215 220

Ser Asp Tyr Glu Ala Leu Arg Ser Ala Phe Val Val Glu His Tyr Pro

225 230 235 240

Glu Lys Pro Asp Ile Asp Phe His Lys Tyr Met Thr Arg Ala Val Glu

245 250 255

Tyr Glu Phe Lys Arg Val Val Gly Ile Ser Trp Tyr Leu Trp Leu Phe

260 265 270

Val Ile Leu Phe Leu Leu Leu Asn Ile Asn Gly Trp His Thr Tyr Phe

275 280 285

Trp Leu Ala Phe Leu Pro Leu Phe Leu Leu Leu Ile Val Gly Ala Lys

290 295 300

Leu Glu His Ile Ile Thr Arg Leu Ala Gln Glu Ala Ala Ile Ser Leu

305 310 315 320

Ser Asn Asn Thr Glu Glu Val Pro Lys Ile Lys Pro Cys Lys Asp His

325 330 335

Phe Trp Phe His Lys Pro Glu Leu Val Leu His Leu Ile His Phe Ile

340 345 350

Leu Phe Gln Asn Ser Phe Glu Ile Ser Phe Phe Phe Trp Ile Leu Val

355 360 365

Ser Glu Gly Phe Gly Ser Cys Met Met Glu Arg Lys Pro Tyr Val Ile

370 375 380

Ser Arg Leu Val Ile Gly Val Ile Ile Glu Val Ile Cys Ser Tyr Ile

385 390 395 400

Thr Leu Pro Leu Tyr Ala Ile Val Thr His Met Thr Gly Gln Ile Lys

405 410 415

Leu His Gly Phe Gly Ser Arg Val His Glu Ser Val His Gly Trp Ile

420 425 430

Gly Leu Arg Lys Lys Pro Phe Ser Phe Trp Lys Ile Pro Gly Gly Asp

435 440 445

Pro Asn Ala Asp Ser Gly Arg Glu Ala Asp Val Thr Arg Arg Val Ala

450 455 460

Lys Glu Arg Ser Gly Ser Ser Arg Ser Met Pro Met Ala Pro Ala Asp

465 470 475 480

Glu Ile Val Thr Val Asp Asp Val Ala Val Ala Ala Ala Ala Val Gly

485 490 495

Gln Gly Pro

13

1798

Zea mays

Mlo8

13

ccgggagcgg caagactggg acacgccagc cctttatttc gagctttctg cgtgcgggag 60

gggggttgac aggcgatggc ggccgagggc gaggcggcgg cgctggagtt cacaccgacg 120

tggatcgtcg cggcggtctg ctctctcatc gtgctcctct cgctcgtcgc cgagcgatgc 180

ctccactacc tcggcaagac gctcaagagg aagaaccaga agccgctctt cgaggcgctg 240

ctcaaggtca aagaagagtt gatgcttctg gggttcatct ccctgctgct gacggtgttc 300

caggggatga tccggaggac gtgcatccct gaacgctgga cattccacat gctgccatgc 360

gagaagccag atgagaaggc cggtgaggcc gccaccatgg agcattttgt agggacgctt 420

ggcaggatcg gtaggcgtct gttgcaggaa ggcactgctg gggctgagca atgccagaag 480

aagggaaaag ttccactttt gtcccttgaa gccatacatc agctgcacat tttcatattt 540

gttctggcaa tcacacatgt tattttcagc gtcacaacta tgcttttagg aggtgcacag 600

atacaccaat ggaaacagtg ggagaatgga attaaaaaag atgctcctgg aaatgggcct 660

aaggtaacca atgtacatca tcatgaattt atcaagaaac gttttaaggg tattggcaaa 720

gattctataa tattgagttg gctgcattct tttggtaagc agttttatag atcagtatct 780

aaatcagatt acaccacaat gcgtcttggt tttatcatga ctcactgccc tggaaatcca 840

aaatttgatt tccatagata catggtaagg gttttagagg cggattttaa gaaagtggta 900

ggcataagct ggtacttgtg ggtcttcgtg gtgatatttc tgttgctgaa tgttaatggc 960

tggcacacat acttttggat tgctttcctt ccccttattc ttctgttagc cattggcact 1020

aagctggagc atgtcatagc tcagctagcc catgatgtag ctgagaagca cacagcggtc 1080

gagggcgatg tgatcgtaaa accatcagat gaacacttct ggttcggcaa gcctagggtt 1140

atcctttacc tgatccactt catcctcttt cagaatgcgt ttgagattgc gtttttcttc 1200

tggatactga gcacttatgg attcgactcg tgcatcatgg gacaagttcg ttttattgtg 1260

ccaaggcttg tcatcggggt ggttattcag cttctctgca gctacagcac cttgcctctg 1320

tatgcaattg taacccagat ggggagctgc tacaagaagg agatcttcaa cgagcatgtg 1380

cagcagggcg tcctgggctg ggctcagaag gtcaagatga aaaagggact gaagggagct 1440

gcatctgcta gcaaggacga atcgattacc aatgccgatt cggcaggacc ttccgttaag 1500

attgaaatgg cgaaggctgg ggaggatgtt gagatcgttg gaaacacagg ttgattggga 1560

caatagggtg cccgtgttgt aatgatgtaa caggttaata tgccatcatc tttttttgta 1620

gatactagat agcttgctgt ggcaataccg caatagcggt gaactagaga aggtgagttt 1680

gggcccggga gcctcatctg ttatcggtcc agtaggaagc aaattcttat atacgggata 1740

tcgataagaa atgaactaag aacatgttcc tggattcatt aaaaaaaaaa aaaaaaaa 1798

14

492

Zea mays

Mlo8

14

Met Ala Ala Glu Gly Glu Ala Ala Ala Leu Glu Phe Thr Pro Thr Trp

1 5 10 15

Ile Val Ala Ala Val Cys Ser Leu Ile Val Leu Leu Ser Leu Val Ala

20 25 30

Glu Arg Cys Leu His Tyr Leu Gly Lys Thr Leu Lys Arg Lys Asn Gln

35 40 45

Lys Pro Leu Phe Glu Ala Leu Leu Lys Val Lys Glu Glu Leu Met Leu

50 55 60

Leu Gly Phe Ile Ser Leu Leu Leu Thr Val Phe Gln Gly Met Ile Arg

65 70 75 80

Arg Thr Cys Ile Pro Glu Arg Trp Thr Phe His Met Leu Pro Cys Glu

85 90 95

Lys Pro Asp Glu Lys Ala Gly Glu Ala Ala Thr Met Glu His Phe Val

100 105 110

Gly Thr Leu Gly Arg Ile Gly Arg Arg Leu Leu Gln Glu Gly Thr Ala

115 120 125

Gly Ala Glu Gln Cys Gln Lys Lys Gly Lys Val Pro Leu Leu Ser Leu

130 135 140

Glu Ala Ile His Gln Leu His Ile Phe Ile Phe Val Leu Ala Ile Thr

145 150 155 160

His Val Ile Phe Ser Val Thr Thr Met Leu Leu Gly Gly Ala Gln Ile

165 170 175

His Gln Trp Lys Gln Trp Glu Asn Gly Ile Lys Lys Asp Ala Pro Gly

180 185 190

Asn Gly Pro Lys Val Thr Asn Val His His His Glu Phe Ile Lys Lys

195 200 205

Arg Phe Lys Gly Ile Gly Lys Asp Ser Ile Ile Leu Ser Trp Leu His

210 215 220

Ser Phe Gly Lys Gln Phe Tyr Arg Ser Val Ser Lys Ser Asp Tyr Thr

225 230 235 240

Thr Met Arg Leu Gly Phe Ile Met Thr His Cys Pro Gly Asn Pro Lys

245 250 255

Phe Asp Phe His Arg Tyr Met Val Arg Val Leu Glu Ala Asp Phe Lys

260 265 270

Lys Val Val Gly Ile Ser Trp Tyr Leu Trp Val Phe Val Val Ile Phe

275 280 285

Leu Leu Leu Asn Val Asn Gly Trp His Thr Tyr Phe Trp Ile Ala Phe

290 295 300

Leu Pro Leu Ile Leu Leu Leu Ala Ile Gly Thr Lys Leu Glu His Val

305 310 315 320

Ile Ala Gln Leu Ala His Asp Val Ala Glu Lys His Thr Ala Val Glu

325 330 335

Gly Asp Val Ile Val Lys Pro Ser Asp Glu His Phe Trp Phe Gly Lys

340 345 350

Pro Arg Val Ile Leu Tyr Leu Ile His Phe Ile Leu Phe Gln Asn Ala

355 360 365

Phe Glu Ile Ala Phe Phe Phe Trp Ile Leu Ser Thr Tyr Gly Phe Asp

370 375 380

Ser Cys Ile Met Gly Gln Val Arg Phe Ile Val Pro Arg Leu Val Ile

385 390 395 400

Gly Val Val Ile Gln Leu Leu Cys Ser Tyr Ser Thr Leu Pro Leu Tyr

405 410 415

Ala Ile Val Thr Gln Met Gly Ser Cys Tyr Lys Lys Glu Ile Phe Asn

420 425 430

Glu His Val Gln Gln Gly Val Leu Gly Trp Ala Gln Lys Val Lys Met

435 440 445

Lys Lys Gly Leu Lys Gly Ala Ala Ser Ala Ser Lys Asp Glu Ser Ile

450 455 460

Thr Asn Ala Asp Ser Ala Gly Pro Ser Val Lys Ile Glu Met Ala Lys

465 470 475 480

Ala Gly Glu Asp Val Glu Ile Val Gly Asn Thr Gly

485 490

15

1557

Zea mays

Mlo9

15

cattcaccga ggagaaatct gaaaactagt taccataacc atgaaggctg ccgtgaggga 60

tatgagtcat ttgtttcgca tgaaggtttg gagcagctgc accggtttat atttgtaatg 120

gctgtgactc atgtgactta tagctgcctg acgatgttac tagcaatact caagatccat 180

aaatggagaa aatgggagga tgaagcattc agggataatc atgaatcatt ttctcagatc 240

gcatatgagt cagcaactag aaggcaacca gcacttacca aatcctattc attccgctct 300

tggagccaaa ataatgtggt catgtggctt gtttgcttta ttgcgcaatt tggtcagtct 360

gttgttcgag cagactatct tatcctccgc aagggtttta taatgaccca caatcttccg 420

ccaacatatg atttccacaa ttacatgata cgctcaatgg aagaggagtt tgagaagatt 480

gtcggagtga gtggactgtt gtggggcttc gttgttgctt ttatgttatt taatgtagac 540

ggatccaacc tgtacttttg gatagcaatt cttcctgtaa ctcttgttct tctggtgggt 600

gcaaagctgc agcatgtgat tgcgactcta acagcagagg gcgcaaagat gagcacctac 660

gggccaagga tacagccaag ggatgatctc ttttggttca agaaaccaga gtttctcctt 720

tggttgatac acttcgttct ctttcagaat gccttcgagt tggcttcgtt cttctggttc 780

tggtggcaat ttggttatga ttcatgcttc atcaaaaacc accttttggt atattgccgc 840

cttatactgg ggtttgctgg acagttcctg tgcagttaca gtacattacc tgtatatgcg 900

ctggtcactc agatggggtc caagtacaag gctgcgctga tcccgcggag gatcagggag 960

accatacatg ggtggggaaa agcgacgagg aagaagcgcc ggcgccgccg cggcgacgac 1020

tcgacggtcc gcacggagac gagcacggtg tgctcgctca cggacgacga cgaagacttc 1080

gaagacgacg acgaccacca ccaccacgga ccgtcgtacg ataccccgag agccgggggc 1140

cgcccgccgt acctgaagat cgagacgcat cgtcagtcgg gcagcgggca cgacggcccc 1200

aggcccggcg gcaccccgtg cttccacccc agcggcagcg gcagcgggca cgccatgctc 1260

ctgcggcagg cctccgtctc cgcgccgtcg tcgccgtcgt accgcggagg caataacgtg 1320

acaaggtcgg cgtccatgcc cgggatcgcc gcgctgagga caacgggctc cggcacgccg 1380

acgcgcgtga gccatgagga gcctacttga tgcgcgcccg tgcatgccat ggatggcttg 1440

ctagtgcaga cgtactgagg actgagctgc tagtgtagca ccggatttga aggtatatcg 1500

atatatatat acacacgaaa agatttgggg tgacaaaaaa aaaaaaaaaa aaaaaaa 1557

16

469

Zea mays

Mlo9

16

His Ser Pro Arg Arg Asn Leu Lys Thr Ser Tyr His Asn His Glu Gly

1 5 10 15

Cys Arg Glu Gly Tyr Glu Ser Phe Val Ser His Glu Gly Leu Glu Gln

20 25 30

Leu His Arg Phe Ile Phe Val Met Ala Val Thr His Val Thr Tyr Ser

35 40 45

Cys Leu Thr Met Leu Leu Ala Ile Leu Lys Ile His Lys Trp Arg Lys

50 55 60

Trp Glu Asp Glu Ala Phe Arg Asp Asn His Glu Ser Phe Ser Gln Ile

65 70 75 80

Ala Tyr Glu Ser Ala Thr Arg Arg Gln Pro Ala Leu Thr Lys Ser Tyr

85 90 95

Ser Phe Arg Ser Trp Ser Gln Asn Asn Val Val Met Trp Leu Val Cys

100 105 110

Phe Ile Ala Gln Phe Gly Gln Ser Val Val Arg Ala Asp Tyr Leu Ile

115 120 125

Leu Arg Lys Gly Phe Ile Met Thr His Asn Leu Pro Pro Thr Tyr Asp

130 135 140

Phe His Asn Tyr Met Ile Arg Ser Met Glu Glu Glu Phe Glu Lys Ile

145 150 155 160

Val Gly Val Ser Gly Leu Leu Trp Gly Phe Val Val Ala Phe Met Leu

165 170 175

Phe Asn Val Asp Gly Ser Asn Leu Tyr Phe Trp Ile Ala Ile Leu Pro

180 185 190

Val Thr Leu Val Leu Leu Val Gly Ala Lys Leu Gln His Val Ile Ala

195 200 205

Thr Leu Thr Ala Glu Gly Ala Lys Met Ser Thr Tyr Gly Pro Arg Ile

210 215 220

Gln Pro Arg Asp Asp Leu Phe Trp Phe Lys Lys Pro Glu Phe Leu Leu

225 230 235 240

Trp Leu Ile His Phe Val Leu Phe Gln Asn Ala Phe Glu Leu Ala Ser

245 250 255

Phe Phe Trp Phe Trp Trp Gln Phe Gly Tyr Asp Ser Cys Phe Ile Lys

260 265 270

Asn His Leu Leu Val Tyr Cys Arg Leu Ile Leu Gly Phe Ala Gly Gln

275 280 285

Phe Leu Cys Ser Tyr Ser Thr Leu Pro Val Tyr Ala Leu Val Thr Gln

290 295 300

Met Gly Ser Lys Tyr Lys Ala Ala Leu Ile Pro Arg Arg Ile Arg Glu

305 310 315 320

Thr Ile His Gly Trp Gly Lys Ala Thr Arg Lys Lys Arg Arg Arg Arg

325 330 335

Arg Gly Asp Asp Ser Thr Val Arg Thr Glu Thr Ser Thr Val Cys Ser

340 345 350

Leu Thr Asp Asp Asp Glu Asp Phe Glu Asp Asp Asp Asp His His His

355 360 365

His Gly Pro Ser Tyr Asp Thr Pro Arg Ala Gly Gly Arg Pro Pro Tyr

370 375 380

Leu Lys Ile Glu Thr His Arg Gln Ser Gly Ser Gly His Asp Gly Pro

385 390 395 400

Arg Pro Gly Gly Thr Pro Cys Phe His Pro Ser Gly Ser Gly Ser Gly

405 410 415

His Ala Met Leu Leu Arg Gln Ala Ser Val Ser Ala Pro Ser Ser Pro

420 425 430

Ser Tyr Arg Gly Gly Asn Asn Val Thr Arg Ser Ala Ser Met Pro Gly

435 440 445

Ile Ala Ala Leu Arg Thr Thr Gly Ser Gly Thr Pro Thr Arg Val Ser

450 455 460

His Glu Glu Pro Thr

465

17

533

Hordeum vulgare

17

Met Ser Asp Lys Lys Gly Val Pro Ala Arg Glu Leu Pro Glu Thr Pro

1 5 10 15

Ser Trp Ala Val Ala Val Val Phe Ala Ala Met Val Leu Val Ser Val

20 25 30

Leu Met Glu His Gly Leu His Lys Leu Gly His Trp Phe Gln His Arg

35 40 45

His Lys Lys Ala Leu Trp Glu Ala Leu Glu Lys Met Lys Ala Glu Leu

50 55 60

Met Leu Val Gly Phe Ile Ser Leu Leu Leu Ile Val Thr Gln Asp Pro

65 70 75 80

Ile Ile Ala Lys Ile Cys Ile Ser Glu Asp Ala Ala Asp Val Met Trp

85 90 95

Pro Cys Lys Arg Gly Thr Glu Gly Arg Lys Pro Ser Lys Tyr Val Asp

100 105 110

Tyr Cys Pro Glu Gly Lys Val Ala Leu Met Ser Thr Gly Ser Leu His

115 120 125

Gln Leu His Val Phe Ile Phe Val Leu Ala Val Phe His Val Thr Tyr

130 135 140

Ser Val Ile Thr Ile Ala Leu Ser Arg Leu Lys Met Arg Thr Trp Lys

145 150 155 160

Lys Trp Glu Thr Glu Thr Thr Ser Leu Glu Tyr Gln Phe Ala Asn Asp

165 170 175

Pro Ala Arg Phe Arg Phe Thr His Gln Thr Ser Phe Val Lys Arg His

180 185 190

Leu Gly Leu Ser Ser Thr Pro Gly Ile Arg Trp Val Val Ala Phe Phe

195 200 205

Arg Gln Phe Phe Arg Ser Val Thr Lys Val Asp Tyr Leu Thr Leu Arg

210 215 220

Ala Gly Phe Ile Asn Ala His Leu Ser Gln Asn Ser Lys Phe Asp Phe

225 230 235 240

His Lys Tyr Ile Lys Arg Ser Met Glu Asp Asp Phe Lys Val Val Val

245 250 255

Gly Ile Ser Leu Pro Leu Trp Gly Val Ala Ile Leu Thr Leu Phe Leu

260 265 270

Asp Ile Asn Gly Val Gly Thr Leu Ile Trp Ile Ser Phe Ile Pro Leu

275 280 285

Val Ile Leu Leu Cys Val Gly Thr Lys Leu Glu Met Ile Ile Met Glu

290 295 300

Met Ala Leu Glu Ile Gln Asp Arg Ala Ser Val Ile Lys Gly Ala Pro

305 310 315 320

Val Val Glu Pro Ser Asn Lys Phe Phe Trp Phe His Arg Pro Asp Trp

325 330 335

Val Leu Phe Phe Ile His Leu Thr Leu Phe Gln Asn Ala Phe Gln Met

340 345 350

Ala His Phe Val Trp Thr Val Ala Thr Pro Gly Leu Lys Lys Cys Tyr

355 360 365

His Thr Gln Ile Gly Leu Ser Ile Met Lys Val Val Val Gly Leu Ala

370 375 380

Leu Gln Phe Leu Cys Ser Tyr Met Thr Phe Pro Leu Tyr Ala Leu Val

385 390 395 400

Thr Gln Met Gly Ser Asn Met Lys Arg Ser Ile Phe Asp Glu Gln Thr

405 410 415

Ser Lys Ala Leu Thr Asn Trp Arg Asn Thr Ala Lys Glu Lys Lys Lys

420 425 430

Val Arg Asp Thr Asp Met Leu Met Ala Gln Met Ile Gly Asp Ala Thr

435 440 445

Pro Ser Arg Gly Ser Ser Pro Met Pro Ser Arg Gly Ser Ser Pro Val

450 455 460

His Leu Leu His Lys Gly Met Gly Arg Ser Asp Asp Pro Gln Ser Ala

465 470 475 480

Pro Thr Ser Pro Arg Thr Gln Gln Glu Ala Arg Asp Met Tyr Pro Val

485 490 495

Val Val Ala His Pro Val His Arg Leu Asn Pro Asn Asp Arg Arg Arg

500 505 510

Ser Ala Ser Ser Ser Ala Leu Glu Ala Asp Ile Pro Ser Ala Asp Phe

515 520 525

Ser Phe Ser Gln Gly

530

18

30

Artificial Sequence

Description of Artificial Sequence

oligonucleotide primer

18

agttatccaa ggcatgccgg tggtaaaact 30

19

28

Artificial Sequence

Description of Artificial Sequence

oligonucleotide primer

19

aagtcatcga gggaggcatc acttacag 28

20

32

Artificial Sequence

Description of Artificial Sequence

oligonucleotide primer

20

agagaagcca acgccawcgc ctcyatttcg tc 32