EA047146B1 - GENE CONFERING RESISTANCE TO PATHOGENIC FUNGUS - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to a nucleic acid molecule encoding a polypeptide that confers resistance to a plant pathogenic fungus such as Helminthosporium turcicum. The present invention also relates to a plant (or part thereof) containing a nucleic acid molecule, and to methods including the nucleic acid molecule.

Prerequisites for creating the invention

Corn (Zea mays L.) has many pathogenic fungi that cause leaf diseases. A fungus that can cause much more harm in tropical as well as temperate climates such as those found in much of Europe and North America, as well as Africa and India, is known as Helminthosporium turcicum or synonymously Exserohilum turcicum ( teleomorph: Setosphaeria turcica). H. turcicum is the cause of the leaf spot disease known as Northern Corn Leaf Blight (NCLB), which can occur at epidemic rates during wet years, attacking vulnerable corn varieties and causing extensive damage, resulting in significant yield losses that can amount to 30%, and it can affect even wider areas. For this reason, people have been trying to find natural resistance in genetic material since the 1970s. Currently, quantitative and qualitative stability is known, albeit not completely. While oligogenetically or polygenetically inherited quantitative resistance is incomplete and non-race-specific in phenotype and is affected by additional and partially dominant genes, qualitative resistance, in turn, is usually race-specific and can be inherited. through individual, mostly dominant genes at loci such as HT1, HT2, HT3, Html or HTN1 (Lipps et al., 1997, Interaction of Ht and partial resistance to Exserohilum turcicum in maize. Plant Disease 81: 277-282; Welz and Geiger, 2000, Resistance genes to northern corn leaf blight in different maize populations Plant Breeding 119: 1-14). Backcrosses into many commonly used maize inbred lines, such as W22, A619, B37 or B73, have successfully introgressed HT loci where they show partial dominance and expression as a function of the appropriate genetic background (Welz, 1998, Genetics and Epidemiology of the Zea Pathosystem mays / Setosphaeria turcica Habilitationsschrift, Institut fur Pflanzenzuchtung, Saatgutforschung und Population-genetik, Universitat Hohenheim).

Despite this complex genetic architecture of NCLB resistance in maize, until now, mainly the use of the HT1 gene located on the long arm of chromosome 2, together with partial quantitative resistance, has been sufficient to control helminthosporiosis in maize (Welz, 1998). The reason for this is that worldwide, races 0 and 1 of H. turcicum are the most common (approximately 55%) (Lipps et al., 1997), while other races, such as 2N and 23N, are rare even in geographically limited areas (Welz, 1998). This race 0 is avirulent to HT1 maize (see also Table 1) and thus, while endowed with suitable quantitative resistance, exhibits sufficient overall resistance to NCLB. However, many studies have reported increasing dissemination of less common races (Jordan et al., 1983, Occurrence of Exserohilum turcicum race 2 in corn in the central and eastern United States. Plant Disease 67: 1163-1165; Welz, 1998). The reasons for this lie in the population dynamics of the pathogen, which provide changes in the virulence of pathogens due to new mutations in avirulence genes and new combinations of available virulence genes. This can lead to the emergence of new, sometimes more aggressive pathogenic races. In Brazil, for example, the population of H. turcicum already appears to be significantly more diverse in terms of racial composition than, for example, in North America. Already in the 1990s, races of H. turcicum were reported to have overcome the resistance conferred by the HT1 gene. In addition, there is instability of genes for resistance to certain environmental factors, such as temperature and light intensity in some climatic zones (Thakur et al., 1989, Effect of temperature and light on the virulence of Exserohilum turcicum in maize. Phytopathology 1989, 79: 631- 635). As a consequence, the use of new HT resistance genes for the production of commercial maize plants, targeting more complete and longer-lasting resistance to H. turcicum in maize, is increasing in importance.

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Table 1

Review of resistance (R) and sensitivity (S) loci of resistance to different races of Helminthosporium turcicum:

Pathogen Host response (Ht) to each race Race designation Ht HT1 locus HT2 locus NTZ locus HTN1 gene 0 R R R R 1 S R R R 2 R S R R 3 R R S R N R R R S 12 S S R R 23 R S S R 2N R S R S 12N S S R S 23N R S S S 123N S S S S

One source of monogenic resistance of the HTN1 gene is the Mexican Landrace pig Pepitilla (Gevers, 1975, New major resistance gene to Helminthosporium leaf spot of corn. Plant Disease Rep 59: 296-300). Introgression lines of the HTN1 gene show mapping of the gene to the long arm of chromosome 8. Unlike conventional HT resistance genes, the HTN1 gene confers resistance by delaying the onset of sporulation and thus combats the development of lesions. As a result, fewer lesions are produced and the number of sporulation zones is reduced (Simcox and Bennetzen, 1993, Use of molecular markers to study resistance to Setospaeria turcica in maize. Phytopathology 83: 1326-1330). Chlorotic-necrotic lesions, such as those due to resistance conferred by HT1, HT2 or HT3, do not form (Gevers, 1975). WO 2015/032494 discloses the identification of the causative gene, RLK1, conferring the Pepitilla 8.06 bp resistance phenotype in maize, and describes molecular markers suitable for exploiting this resistance locus without tightly linked unwanted linked load resulting in a negative impact on maximum potential. harvest.

WO 2011/163590 disclosed the PH99N and PH26N genotypes as alternative sources for 5 base pair NCLB resistance on chromosome 8.

To identify the NCLB resistance gene from the maize hybrid DK888, in 2010, Chang et al. published a study fine-mapping the 8.06 bp resistance locus (Chang et al. 2010 Characterization and fine mapping of the resistance locus for northern leaf blight). maize for 8.06 bp Theoretical and Applied Genetics 121(2):205-227). Studies of Helminthosporium race specificity initially showed that the resistance locus is functionally closely linked to the HT2 and HTN1 genes. Annotation of the genomes of a 0.46 Mb chromosome fragment using reference B73 hinted at several putative open reading frames; however, the causative gene has not been identified and functional validation has not been described.

It is therefore an object of the present invention to identify and/or further characterize plant resistance genes encoding polypeptides that confer or enhance resistance to a pathogenic fungus such as Helminthosporium turcicum.

Summary of the invention

The present invention provides a nucleic acid molecule containing or consisting of a nucleotide sequence having the nucleotide sequence presented in SEQ ID NO: 1 or SEQ ID NO: 2. Also provided is a nucleic acid molecule containing or consisting of a nucleotide sequence encoding the amino acid sequence represented by in SEQ ID NO: 3.

Also provided is a nucleic acid molecule containing or consisting of a nucleotide sequence having at least 96% identity with the nucleotide sequence presented in SEQ ID NO: 1 or SEQ ID NO: 2, and a nucleic acid molecule containing or consisting of a nucleotide sequence encoding an amino acid sequence having at least 92% identity with the amino acid sequence set forth in SEQ ID NO: 3.

Also proposed is a nucleic acid molecule containing or consisting of a nucleotide sequence that hybridizes with a complementary strand of the nucleotide sequence,

- 2 047146 presented in SEQ ID NO: 1 or SEQ ID NO: 2, or hybridizing with the complementary strand of a nucleotide sequence encoding the amino acid sequence presented in SEQ ID NO: 3, under stringent hybridization conditions.

In addition, a nucleic acid molecule is provided containing or consisting of a nucleotide sequence encoding a protein, wherein said protein is derived from the amino acid sequence encoded by the nucleotide sequence presented in SEQ ID NO: 1 or SEQ ID NO: 2, or from the amino acid sequence represented by in SEQ ID NO: 3, by substitution, deletion and/or addition of at least one amino acid.

The nucleic acid molecule of the present invention encodes a polypeptide that is wall-associated receptor-like kinase 1 (WAK RLK1), or functionally belongs to the family of wall-associated receptor-like kinases.

The nucleic acid molecule of the present invention encodes a polypeptide capable of conferring (or increasing) resistance to a plant disease caused by a pathogenic fungus, in particular Helminthosporium turcicum, in a preferred embodiment of the present invention, Helminthosporium turcicum race 0, 1 and/or N in a plant, in a preferred embodiment of the present invention, in a plant of the species Zea mays in which the polypeptide is expressed.

In another aspect, a vector or expression cassette comprising a nucleic acid molecule of the present invention is provided. In a preferred embodiment of the present invention, in a vector or expression cassette, a nucleotide sequence of the present invention is operably linked to a regulatory element that causes the nucleotide sequence to be expressed in a plant cell.

Also provided is a host cell containing a nucleic acid molecule of the present invention or a vector or expression cassette of the present invention.

In another aspect, a polypeptide encoded by a nucleic acid molecule of the present invention is provided. The polypeptide is wall-associated receptor-like kinase 1 (WAK RLK1), or functionally belongs to the wall-associated receptor-like kinase 1 family. In a preferred embodiment of the present invention, the polypeptide is capable of conferring (or increasing) resistance to a plant disease caused by Helminthosporium turcicum, in a preferred embodiment of the present invention Helminthosporium turcicum race 0, 1 and/or N in the plant, in a preferred embodiment of the present invention in plants of the Zea mays species in which the polypeptide is expressed.

In addition, the polypeptide may be capable of conferring or increasing resistance to plant disease caused by Helminthosporium turcicum race 2 and/or 3 in the plant, in a preferred embodiment of the present invention, the Zea mays plant in which the polypeptide is expressed. The plant may show a sensitive response to infection by Helminthosporium turcicum races 2 and/or 3.

In addition, a plant is provided, in a preferred embodiment of the present invention, a plant of the Zea mays species, containing a nucleic acid molecule of the present invention transgenically (as a transgene) or endogenously (as an endogenous gene), a vector of the present invention, an expression cassette of the present invention, or a polypeptide according to the present invention. In a preferred embodiment of the present invention, the plant is resistant to plant disease caused by Helminthosporium turcicum, in particular Helminthosporium turcicum races 0, 1 and/or N. The plant may show a sensitive response to infection by Helminthosporium turcicum races 2 and/or 3. is a transgenic plant or a genetically edited plant. The plant may be a plant endogenously containing a nucleic acid molecule of the present invention, wherein the flanking regions of the genome do not contain an interval derived from A619HT2 or A619HT3 located between the alleles of the SYN14136 marker and the MA0021 marker, or an interval derived from A619HT2 or A619HT3 located between the alleles of the MA0022 marker and the SYN4196 marker. Also provided is a part of a plant of the present invention, a plant cell of a plant of the present invention, and a seed of a plant of the present invention, wherein the seed contains a nucleic acid molecule of the present invention transgenically (as a transgene) or endogenously (as an endogenous gene), a vector of the present invention, an expression cassette of the present invention or a polypeptide of the present invention.

According to another aspect, there is provided a method or method for identifying or selecting a plant, in a preferred embodiment of the present invention, a plant of the species Zea mays, having increased resistance to a plant disease caused by a pathogenic fungus, in a preferred embodiment of the present invention, Helminthosporium turcicum, more in In a preferred embodiment of the present invention, Helminthosporium turcicum race 0, 1 and/or N, or a part, cell or seed thereof, comprising the following steps: (a) determination in the plant or part, cell or seed (or in a sample of the plant, or its part, cell or seed), the presence of a nucleic acid molecule of the present invention as described above, or a nucleic acid molecule containing or consisting of a nucleotide sequence selected from

- 3 047146 group consisting of: (i) a nucleotide sequence having at least 60% identity with the nucleotide sequence presented in SEQ ID NO: 1 or SEQ ID NO: 2, (ii) a nucleotide sequence encoding an amino acid sequence having at least 60% identity with the amino acid sequence presented in SEQ ID NO: 3, (iii) a nucleotide sequence having at least 85% identity with nucleotide positions 1-920 of the nucleotide sequence, presented in SEQ ID NO: 1 or SEQ ID NO: 2 (exon 1; SEQ ID NO: 6), or having at least 60% identity with nucleotide positions 23252-23288 of the nucleotide sequence presented in SEQ ID NO : 1 (exon 2; SEQ ID NO: 7), or with nucleotide positions 921-957 of the nucleotide sequence presented in SEQ ID NO: 2 (exon 2; SEQ ID NO: 7), and/or having at least 98% -identity with nucleotide positions 23586-24632 of the nucleotide sequence presented in SEQ ID NO: 1 (exon 3; SEQ ID NO: 8), or at nucleotide positions 958-2004 of the nucleotide sequence shown in SEQ ID NO: 2 (exon 3; SEQ ID NO: 8), (iv) a nucleotide sequence encoding an amino acid sequence having at least 75 % identity with positions 1-306 of the amino acid sequence presented in SEQ ID NO: 3, and/or having at least 60% identity with positions 307-319 of the amino acid sequence presented in SEQ ID NO: 3, and/ or having at least 98% identity to positions 320-668 of the amino acid sequence set forth in SEQ ID NO: 3, wherein the nucleic acid molecule of any one of clauses (i) to (iv) encodes a polypeptide capable of imparting or enhancing resistance to a plant disease caused by a pathogenic fungus, in a preferred embodiment of the present invention - Helminthosporium turcicum, in a more preferred embodiment of the present invention - Helminthosporium turcicum race 0, 1 and/or N, in a plant, in a preferred embodiment of the present invention - in plant of the Zea mays species in which the polypeptide is expressed, and wherein, in a preferred embodiment of the present invention, the nucleic acid molecule of any one of (i)(iv) encodes a polypeptide that is or functionally belongs to the receptor-like kinase 1 (WAK) family RLK1), associated with the wall; and (b) identifying or selecting a plant in which, or in a part, cell or seed of which, a nucleic acid molecule as defined in paragraph (a) is present as having resistance or increased resistance to the plant disease caused by a pathogenic fungus, in a preferred embodiment of the present invention - Helminthosporium turcicum, in a more preferred embodiment of the present invention - Helminthosporium turcicum races 0, 1 and/or N.

Also provided is a method or method for identifying a plant, in a preferred embodiment of the present invention, a plant of the species Zea mays, which is resistant to the plant disease caused by Helminthosporium turcicum, in particular Helminthosporium turcicum race 0, 1 and/or N, and it contains a nucleic acid molecule of the present invention is endogenous, wherein the method or method comprises identifying in plant alleles at least two markers, wherein at least one of said markers is located on or within the chromosomal interval between SYN14136 and the nucleic acid molecule of the present invention, and at least one of said markers is located on or within a chromosomal interval between the nucleic acid molecule of the present invention and SYN4196.

In another aspect, a plant identified or selected by the identification or selection method or method of the present invention, or a progeny thereof, is provided.

In another aspect, a method is provided for identifying a resistance gene allele that confers or increases resistance to a plant disease caused by a pathogenic fungus, in a preferred embodiment of the present invention - Helminthosporium turcicum, in a more preferred embodiment of the present invention - Helminthosporium turcicum race 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, in Zea mays and in a preferred embodiment of the present invention encoding a polypeptide that is or functionally belongs to the family of receptor-like kinase 1 (WAK RLK1) associated with a wall, wherein the method comprises the following steps: (a) performing a sequence comparison using (i) at least one coding nucleotide sequence derived from or derived from a Zea mays genotype, wherein the nucleotide sequence, in a preferred embodiment of the present invention, corresponds to the 8.05 bp resistance locus or the 8.06 bp resistance locus, and (ii), as a reference sequence, the nucleotide sequence of the present invention, in a preferred embodiment of the present invention, the nucleotide sequence presented in SEQ ID NO: 1 or SEQ ID NO: 2, or a nucleotide sequence encoding the amino acid sequence presented in SEQ ID NO: 3, or a part thereof, or a consensus sequence derived from a set of at least two nucleotide sequences, wherein , one nucleotide sequence is the nucleotide sequence of the present invention, in a preferred embodiment of the present invention, the nucleotide sequence represented by

- 4 047146 in SEQ ID NO: 1 or SEQ ID NO: 2, or a nucleotide sequence encoding the amino acid sequence presented in SEQ ID NO: 3, or part thereof, and, wherein each nucleotide sequence of the set is at least two nucleotide sequences encodes a polypeptide capable of conferring or increasing resistance to a plant disease caused by a pathogenic fungus, in a preferred embodiment of the present invention - Helminthosporium turcicum, in a more preferred embodiment of the present invention Helminthosporium turcicum race 0, 1 or N, in Zea mays, in which the polypeptide is expressed, and, in a preferred embodiment of the present invention, corresponds to an 8.05 base pair resistance locus or an 8.06 base pair resistance locus, and, in a preferred embodiment of the present invention, encodes a polypeptide that is or functionally belongs to wall-associated receptor-like kinase 1 (WAK RLK1) family, and (b) identification of the allele if sequence comparison reveals (i) nucleotide-level sequence identity of at least 85% identity to nucleotide positions of 1 -920 nucleotide sequence presented in SEQ ID NO: 1 or SEQ ID NO: 2, and/or having at least 60% identity with nucleotide positions 2325223288 of the nucleotide sequence presented in SEQ ID NO: 1, or with positions of nucleotides 921-957 of the nucleotide sequence presented in SEQ ID NO: 2, and/or having at least 98% identity with positions of nucleotides 23586-24632 of the nucleotide sequence presented in SEQ ID NO: 1, or with positions nucleotides 958-2004 of the nucleotide sequence presented in SEQ ID NO: 2, and/or (ii) encoded amino acid level sequence identity constituting at least 75% identity with positions 1-306 of the amino acid sequence presented in SEQ ID NO: 3, and/or having at least 60% identity with positions 307-319 of the amino acid sequence shown in SEQ ID NO: 3, and/or having at least 98% identity with positions 320-668 amino acid sequence shown in SEQ ID NO: 3.

Also proposed is a nucleic acid molecule containing or consisting of a nucleotide sequence of a resistance gene allele identified by a method for identifying a resistance gene allele.

In another aspect, a method is provided for imparting or increasing resistance to a plant disease caused by a pathogenic fungus, in a preferred embodiment of the present invention - Helminthosporium turcicum, in a more preferred embodiment of the present invention - Helminthosporium turcicum race 0, 1, 2, 3, N, 12 , 23, 2N, 12N, 23N and/or 123N, in a plant, in a preferred embodiment of the present invention in a plant of the species Zea mays, comprising the following steps: (a) introducing into at least one cell of the plant a nucleic acid molecule of the present invention including, for example, a nucleic acid molecule containing or consisting of the nucleotide sequence of an identified allele, an expression cassette of the present invention, or a vector of the present invention, (b) regenerating a plant from at least one cell, and (c) causing expression of the nucleic acid molecule in plants.

In another aspect, a method is provided for imparting or increasing resistance to a plant disease caused by a pathogenic fungus, in a preferred embodiment of the present invention - Helminthosporium turcicum, in a more preferred embodiment of the present invention - Helminthosporium turcicum race 0, 1, 2, 3, N, 12 , 23, 2N, 12N, 23N and/or 123N, in a plant of the present invention, comprising the step of reducing the level of expression of a nucleic acid molecule of the present invention, including, for example, a nucleic acid molecule containing or consisting of the nucleotide sequence of the identified allele, expression cassette of the present invention or a vector of the present invention, in a plant or at least one cell of a plant, in a preferred embodiment of the present invention, compared to the level of endogenous gene expression in a resistant wild-type plant.

In another aspect, a method is provided for producing a plant, in a preferred embodiment of the present invention, a plant of the species Zea mays, having (increased) resistance to a plant disease caused by a pathogenic fungus, in a preferred embodiment of the present invention, Helminthosporium turcicum, in a more preferred embodiment of the present invention - Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, or parts thereof, cells or seeds, containing the following steps: (a) introduction into the plant or, according to into at least one plant cell, a nucleic acid molecule of the present invention, including, for example, a nucleic acid molecule containing or consisting of the nucleotide sequence of an identified allele, an expression cassette of the present invention, or a vector of the present invention, (b) optionally regenerating the plant at least at least from one cell and (c) causing expression of a nucleic acid molecule in the plant.

In another aspect, a plant produced by the plant production methods of the present invention, or a progeny thereof, is provided.

A method of combating infection by a pathogenic fungus is also proposed, preferably

- 5 047146 in an embodiment of the present invention - Helminthosporium turcicum, in a more preferred embodiment of the present invention - Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, in a plant population, in a preferred embodiment of the present invention, plants of the species Zea mays, comprising the following steps: (a) growing plants of the present invention in agricultural and horticultural areas and (b) causing expression of a nucleic acid molecule of the present invention, including, for example, a nucleic acid molecule, containing or consisting of the nucleotide sequence of an identified allele, expression cassette of the present invention, or vector of the present invention, in plants.

Another aspect of the present invention is an oligonucleotide having a length of at least 15, 16, 17, 18, 19 or 20, in a preferred embodiment of the present invention at least 21, 22, 23, 24 or 25, more in a preferred embodiment of the present invention, at least 30, 35, 40, 45, 50, 100, 200, 300 or 500 nucleotides, wherein the oligonucleotide is capable of hybridizing or annealing to (i) a nucleic acid molecule of the present invention, (ii ) a nucleic acid molecule containing the nucleotide sequence presented in SEQ ID NO: 4 or SEQ ID NO: 5, or with (iii) a nucleic acid molecule complementary to (i) or (ii).

Also provided is a pair of oligonucleotides or a set containing said oligonucleotides, wherein the oligonucleotides are suitable for annealing as a forward primer and a reverse primer to a region in the plant genome, in a preferred embodiment of the present invention, in the Zea mays genome, which exhibits cosegregation, in a preferred embodiment implementation of the present invention - ideal cosegregation with the nucleic acid molecule of the present invention.

In another aspect, the use of at least one oligonucleotide as a molecular marker or part thereof is provided in a method for identifying or selecting a plant, in a preferred embodiment of the present invention, a plant of the Zea mays species having increased resistance to a plant disease caused by a pathogenic fungus, in a preferred embodiment in an embodiment of the present invention - Helminthosporium turcicum, in a more preferred embodiment of the present invention - Helminthosporium turcicum race 0, 1 and/or N, or a part, cell or seed thereof, or in a method for identifying an allele of a resistance gene that confers or increases resistance to a plant disease caused by a pathogenic fungus, in a preferred embodiment of the present invention - Helminthosporium turcicum, in a more preferred embodiment of the present invention - Helminthosporium turcicum race 0, 1 and/or N, in Zea mays, wherein the molecular marker is capable of detecting at least one single nucleotide polymorphism, to diagnose a deletion or insertion for a nucleic acid molecule of the present invention and/or contains an oligonucleotide as described above and/or is a pair of oligonucleotides or a set as described above.

In one aspect, the use of at least one oligonucleotide or at least two oligonucleotides as a molecular marker or part thereof is provided in a method or method for identifying a plant as described above, or in a method or method for eliminating cohesive cargo closely associated with a molecule nucleic acid of the present invention, to determine the presence or absence of an interval derived from A619HT2 or A619HT3 located between the alleles of the SYN14136 marker and the MA0021 marker, or an interval derived from A619HT2 or A619HT3 located between the alleles of the MA0022 marker and the SYN4196 marker.

In another aspect, a method is provided for determining the presence or absence of a nucleic acid molecule of the present invention, including, for example, a nucleic acid molecule containing or consisting of the nucleotide sequence of an identified allele, in a plant, in a preferred embodiment of the present invention, a plant of the species Zea mays, comprising the steps of: (a) isolating DNA from at least one plant cell and (b) using a molecular marker to determine the presence or absence of a nucleic acid molecule of the present invention, wherein the molecular marker is capable of detecting at least one single nucleotide polymorphism , perform deletion or insertion diagnostics for a nucleic acid molecule of the present invention and/or contains an oligonucleotide as described above and/or is an oligonucleotide pair or set as described above.

Brief description of drawings

In fig. Figure 1 shows the p7U-nativeHT2_CDS_2 vector, which can be used to transform Zea mays plants. An expression cassette containing cDNA of the HT2 gene (SEQ ID NO: 2) under the control of a native promoter (SEQ ID NO: 4) and terminator (SEQ ID NO: 5) was transformed into a binary vector containing, for example, a herbicide resistance gene (for example : BASTA resistance, glyphosate resistance or ALS inhibitor resistance) for subsequent transformation into Agrobacterium tumefaciens for Agrobacterium-mediated plant transformation into maize (Zea mays) genotype A188.

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In fig. 2 A-D shows a multiple CLUSTAL O (1.2.4) sequence alignment of the RLK1 HT2 (SEQ ID NO: 2), HTN1 (SEQ ID NO: 9), PH99N (SEQ ID NO: 11) and PH26N (SEQ ID NO: 11) alleles.

13). Positions with sequence differences are indicated by a missing asterisk in the bottom line of each block.

In fig. 3A and B show multiple sequence alignment of ClustalV (PAM250) RLK1 polypeptides. RLKl_A619HT2_CDS.seq (SEQ ID NO: 3), RLKl_A619HT3_CDS.seq (identical to SEQ ID NO: 3), RLKl_B37HTN_CDS.seq (SEQ ID NO: 10), RLKl_A619HT2_EkZon1.seq (SEQ ID NO: 6), RLKl_A619HT2_ exon2.seq (SEQ ID NO: 7), and RLKl_A619HT2_exon3.seq (SEQ ID NO: 8).

In fig. 4 A-C shows a sequence alignment of the cDNA of the HT2 gene (SEQ ID NO: 2) and the cDNA of the RLK1 allele derived from the A188 genotype. At position 1458-1459 of the A188 allele, a 2-bp AC insertion (white letters on a black background) was identified that causes an early stop codon (in bold and underlined).

In fig. Figure 5 shows the alignment of the amino acid sequences of A619HT2 and the modified A188 RLK1. The modified A188 RLK is 99.1% identical to the A619NT2.

In fig. 6 shows a map of marker positions with reference to AGPv02 marker positions.

Detailed Description of the Invention

The present invention is based on the identification of the HT2 gene, which encodes a polypeptide that imparts (or increases) resistance to a plant against a pathogenic fungus such as Helminthosporium turcicum.

Accordingly, in one aspect of the present invention, there is provided a nucleic acid molecule comprising or consisting of a nucleotide sequence encoding said HT2 gene. In one embodiment of the present invention, the nucleic acid molecule contains or consists of a nucleotide sequence having the nucleotide sequence set forth in SEQ ID NO: 1 (HT2 gene genomic DNA) or SEQ ID NO: 2 (HT2 gene cDNA). Also provided is a nucleic acid molecule containing or consisting of a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 3 (HT2 protein).

Also provided is a nucleic acid molecule containing or consisting of a nucleotide sequence having at least 96% identity with the nucleotide sequence presented in SEQ ID NO: 1 or SEQ ID NO: 2, in a preferred embodiment of the present invention - by the entire length. In a preferred embodiment of the present invention, a nucleotide sequence having at least 97%, 98%, 99% or 99.5% identity with the nucleotide sequence presented in SEQ ID NO: 1 or SEQ ID NO: 2, in the preferred embodiment of the present invention - along the entire length. Also provided is a nucleic acid molecule containing or consisting of a nucleotide sequence encoding an amino acid sequence having at least 92% identity with the amino acid sequence presented in SEQ ID NO: 2, in a preferred embodiment of the present invention - along its entire length . In a preferred embodiment of the present invention, the nucleic acid molecule contains or consists of a nucleotide sequence encoding an amino acid sequence having at least 93%, 94%, 95%, 96%, 97% 98%, 99% or 99.5% identity with the amino acid sequence presented in SEQ ID NO: 3, in a preferred embodiment of the present invention - along the entire length.

In another embodiment of the present invention, there is provided a nucleic acid molecule comprising or consisting of a nucleotide sequence hybridizing to the complementary strand of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, or a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 3, under stringent hybridization conditions.

In addition, a nucleic acid molecule is provided containing or consisting of a nucleotide sequence encoding a protein derived from a protein encoded by the nucleotide sequence SEQ ID NO: 1 or SEQ ID NO: 2, or derived from the amino acid sequence presented in SEQ ID NO: 3 , by substitution, deletion and/or addition of at least one amino acid.

Deletion, substitution or addition of amino acid(s) can be accomplished by a method known in the art. Mutagenesis in a nucleotide sequence can be caused by a known method such as the Kunkel method, the gap duplexing method, or a method similar to such a known method. For example, mutagenesis can be caused by using a mutagenesis kit (for example, Mutant-K or Mutant-G (product name of TAKARA Bio)) based on the site-directed mutagenesis method, LA PCR In Vitro Sequence Mutagenesis Kit (name product from TAKARA Bio) or the like. In an alternative embodiment of the present invention, the mutagenesis method may be a method that uses a chemical mutagen represented by EMS (ethyl methanesulfonate), 5-bromouracil, 2-aminopurine, hydroxylamine, H-methyl-'N'-nitro-'H-7 047146 nitrosoguanidine or other carcinogenic compound, a method comprising radiation treatment using radioactive rays such as X-rays, alpha rays, beta rays, gamma rays or an ion beam, or a method comprising ultraviolet treatment.

When an amino acid residue is changed, the amino acid is, in a preferred embodiment of the present invention, mutated to another amino acid(s) that retains the properties of the amino acid. Examples of amino acid properties are: hydrophobic amino acids (A, I, L, M, F, P, W, Y and V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S and T), amino acids containing aliphatic side chains (G, A, V, L, I and P), amino acids containing hydroxyl groups in the side chains (S, T and Y), amino acids containing sulfur-containing side chains (C and M) , amino acids containing carboxylic acids and amides in side chains (D, N, E and Q), amino acids containing basic side chains (R, K and H), and amino acids containing aromatic side chains (H, F, Y and W ) (amino acids are represented by single-letter codes in parentheses). Amino acid substitutions within each group are called conservative substitutions. Conservative substitutions are preferred.

In a preferred embodiment of the present invention, the nucleic acid molecule of the present invention encodes a polypeptide capable of conferring (or increasing) resistance to a plant disease caused by a pathogenic fungus in a plant in which the polypeptide is expressed.

In addition, the nucleic acid molecule of the present invention encodes a polypeptide that may not be capable of conferring resistance to the plant disease caused by Helminthosporium turcicum race 2 and/or 3 in a plant, in a preferred embodiment of the present invention, in a plant of the species Zea mays, in which polypeptide is expressed. The plant may show a sensitive response to infection by Helminthosporium turcicum races 2 and/or 3.

Another example of a nucleic acid molecule of the present invention may be a nucleic acid molecule that contains or consists of a nucleotide sequence set forth in SEQ ID NO: 23, or a nucleic acid molecule that contains or consists of a nucleotide sequence encoding an amino acid sequence set forth in SEQ ID NO: 24.

In one embodiment of the present invention, the pathogenic fungus belongs to the phylum Ascomycota or Basidiomycota. The pathogenic fungus may belong to the family Pleosporaceae, Pucciniaceae or Botryosphaeriaceae. In a preferred embodiment of the present invention, the pathogenic fungus belongs to the genus Setosphaeria, Bipolaris, Puccinia or Diplodia, in a more preferred embodiment of the present invention, it is the species Helminthosporium turcicum, Setosphaeria rostrata, Setosphaeria glycinea, Setosphaeria holmii, Setosphaeria khartoumensis, Setosphaeria minor, Setosphaeria monoceras , Setosphaeria pedicellata, Setosphaeria prolata, Bipolaris australis, Bipolaris brizae, Bipolaris biich^s. Bipolaris cactivora, Bipolaris clavata, Bipolaris coicis, Bipolaris colocasiae, Bipolaris crotonis, Bipolaris crustacean, Bipolaris cylindrical, Bipolaris euchlaenae, Bipolaris halepensis, Bipolaris heveae, Bipolaris incurvata, Bipolaris indica, Bipolaris iridis, Bipolaris leersiae, polaris micropus, Bipolaris miyakei, Bipolaris multiformis , Bipolaris nicotiae, Bipolaris novae-zelandiae, Bipolaris ovariicola, Bipolaris panici-miliacei, Bipolaris papendorfli, Bipolaris sacchari, Bipolaris salkadehensis, Bipolaris sorghicola, Bipolaris subpapendorfii, Bipolaris tropicalis, Bipolaris urochloae, Bipolaris zeae, asparagi, Puccinia graminis, Puccinia horiana, Puccinia mariae-wilsoniae, Puccinia poarum, Puccinia psidii, Puccinia recondite, Puccinia sessilis, Puccinia sorghi, Puccinia striiformis, Puccinia triticina, Diplodia maydis, Diplodia seriata or Stenocarpella (Diplodia) macrospora, in the most preferred embodiment of the present invention, is Helminthosporium turcicum, Puccinia sorghi, Diplodia macrospora or Bipolaris maydis.

In one embodiment of the present invention, the plant disease is a fungal disease. In a preferred embodiment of the present invention, the plant disease is selected from the group consisting of Northern corn leaf blight (caused by Helminthosporium turcicum), Southern corn leaf blight (caused by Bipolaris maydis), Leaf rust (caused by Puccinia sorghi) and Diplodia (caused by Diplodia macrospora, also called Stenocarpella macrospora). In the most preferred embodiment of the present invention, the plant disease is Northern Corn Leaf Blight (NCLB).

In a preferred embodiment of the present invention, the nucleic acid molecule of the present invention encodes a polypeptide capable of conferring (or increasing in a plant) resistance to northern corn leaf blight (NCLB), that is, resistance to Helminthosporium turcicum, in particular resistance to Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, or resistance to Helminthosporium turcicum races 0, 1 and/or N.

In one embodiment of the present invention, the plant is Hordeum vulgare, Hordeum bulbusom, Sorghum bicolor, Saccharum officinarium, Zea mays, Setaria italica, Oryza minuta, Oriza sativa, Oryza australiensis, Oryza alta, Triticum aestivum, Triticum durum, Secale cereale, Triticale , Malus domestica, Brachypodium distachyon, Hordeum marinum, Aegilops tauschii, Daucus glochidiatus,

- 8 047146

Beta vulgaris, Daucus pusillus, Daucus muricatus, Daucus carota, Eucalyptus grandis, Nicotiana sylvestris, Nicotiana tomentosiformis, Nicotiana tabacum, Nicotiana benthamiana, Solarium lycopersicum, Solarium tuberosum, Coffea canephora, Vitis vinifera, Erythrante guttata, Genlisea aurea , Cucumis sativus, Morus notabilis , Arabidopsis arenosa, Arabidopsis lyrata, Arabidopsis thaliana, Crucihimalaya himalaica, Crucihimalaya wallichii, Cardamine flexuosa, Lepidium virginicum, Capsella bursa pastoris, Olmarabidopsis pumila, Arabis hirsute, Brassica napus, Brassica oleracea, Brassica rapa, Raphanus sativus, Brassica juncacea Brassica nigra Eruca vesicaria subsp. sativa, Citrus sinensis, Jatropha curcas, Populus trichocarpa, Medicago truncatula, Cicer yamashitae, Cicer bijugum, Cicer arietinum, Cicer reticulatum, Cicer judaicum, Cajanus cajanifolius, Cajanus scarabaeoides, Phaseolus vulgaris, Glycine max, Gossypium sp., Astragalus sinicus, Lotus s , Torenia fournieri, Allium sulfur, Allium fistulosum, Allium sativum, Helianthus annuus, Helianthus tuberosus and Allium tuberosum, or any variety or subspecies belonging to one of the above plants, in a more preferred embodiment of the present invention - Hordeum vulgare, Hordeum bulbusom, Sorghum bicolor, Zea mays, Setaria italica, Oryza minuta, Oriza sativa, Oryza australiensis, Oryza alta, Triticum aestivum, Triticum durum, Secale cereale, Triticale, Hordeum marinum, Aegilops tauschii, or any variety or subspecies belonging to one of the above-mentioned plants, or in the most preferred embodiment of the present invention, Hordeum vulgare, Sorghum bicolor, Zea mays, Triticum aestivum, Secale cereale, or any variety or subspecies belonging to one of the above plants.

In a preferred embodiment of the present invention, the plant of the present invention is Zea mays.

In a preferred embodiment of the present invention, the nucleic acid molecule of the present invention encodes a polypeptide that is wall-associated receptor-like kinase 1 (WAK RLK1), or functionally belongs to the wall-associated receptor-like kinase 1 family.

In another aspect, a vector is provided containing a nucleic acid molecule of the present invention. The vector may be a plasmid, cosmid, bacteriophage or expression vector, transformation vector, bifunctional vector or cloning vector, it may be double-stranded or single-stranded, linear or circular, or it may be a prokaryotic or eukaryotic host into whose genome it is introduced either through integration, or through extrachromosomal transformation.

Also provided is an expression cassette containing a nucleic acid molecule of the present invention.

In one embodiment of the present invention, in a vector or expression cassette, a nucleotide sequence of the present invention is operably linked to a regulatory element that causes the nucleotide sequence to be expressed in a plant cell. A plant cell may be susceptible to infection by a pathogenic fungus or be infected by a pathogenic fungus. In a preferred embodiment of the present invention, the plant cell is located in a leaf or leaf tissue. The regulatory element may be a promoter (native, synthetic, core promoter, or chimeric promoter), terminator, enhancer, or cis-acting element. In addition, the regulatory element may be heterologous to a nucleotide sequence operably linked to the regulatory element.

In a preferred embodiment of the present invention, the nucleic acid molecule of the present invention is operably linked in an expression vector to at least one regulatory sequence that allows transcription and optional expression in a prokaryotic or eukaryotic host cell. By way of example, the nucleotide sequence may be under the control of a suitable promoter or terminator. Suitable promoters may be promoters that are constitutively induced (see, for example, the 35S promoter from cauliflower mosaic virus (Odell JT, Nagy F, Chua NH (1985) Identification of DNA sequences required for activity cauliflower mosaic virus 35S promoter. Nature 313, 810-812, 1985); other examples are the Oryza sativa actin promoter (SEQ ID NO: 43) or the Brachypodium distachyon EF1 promoter (SEQ ID NO: 44). , which are pathogen-inducible (see, for example, the PR1 promoter from parsley (Rushton P.J., Torres J.T., Parniske M., Wernert P., Halbrock K. and Somsich I.E. (1996) Elicitor- induced DNA-binding proteins with elicitor response elements in parsley PR1 gene promoters EMBO J. 15(20): 5690-5700). Particularly suitable pathogen-inducible promoters are synthetic or chimeric promoters that do not occur in nature, which consist of several elements, and also contain a minimal promoter, and upstream of the minimal promoter they have at least one cis-regulatory element that acts as a binding site for specific transcription factors. Chimeric promoters are specially designed and induced by various factors or re-primed. Examples of such promoters can be found in WO 2000/29592 and WO 2007/147395. An example of a suitable terminator is

- 9 047146 nopaline synthase terminator (Depicker A, Stahel S, Dheze P, Zambriski P, Goodman HM (1982) Nopaline synthase: mapping the transcript and DNA sequence. J Mol Appl Genet. 1(6) : 561-73).

Also provided is a host cell containing a nucleic acid molecule of the present invention or a vector of the present invention or an expression cassette of the present invention.

The vector or expression cassette may, for example, be introduced into a host cell by conjugation, mobilization, biolistic transformation, Agrobacterium-mediated transformation, transfection, transduction, vacuum infiltration, or electroporation. Methods of this type, as well as methods for preparing the described vectors, are known to one skilled in the art (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory, 3rd edition, 2001).

In one embodiment of the present invention, the host cell may be a prokaryotic cell (eg, a bacterial cell). In one embodiment of the present invention, the host cell may be a eukaryotic cell (eg, a plant cell or yeast cell). Particularly preferred bacterial host cells are Agrobacterium tumefaciens, A. rhizogenes and E. coll.

In another aspect, a protein encoded by a nucleic acid molecule of the present invention is provided.

In another aspect, a plant is provided comprising a nucleic acid molecule of the present invention, a vector of the present invention, an expression cassette of the present invention, or a protein of the present invention. The plant may be a transgenic plant or a genetically edited plant. Also provided are: a plant part of the present invention, a plant cell of a plant of the present invention, and a seed of a plant of the present invention, wherein the seed contains a nucleic acid molecule of the present invention transgenically or endogenously, a vector of the present invention, an expression cassette of the present invention, or a polypeptide of the present invention. the present invention.

From WO 2015/032494, which examined and used introgression lines with Htnl from Pepitilla; This resistance locus is known to be closely linked to genomic regions carrying linkage loads that lead to negative effects on one or more agronomic traits. The first study of the flanking region of HT2 showed that this or similar linked cargo is present not only in the Pepitilla donor for HtN1 introgression, but also in other donors for the Helminthosporium resistance locus A619. In particular, linkage cargo, as part of HT2 introgression, may influence differences in flowering time, an important agronomic trait. This can directly and significantly affect the yield potential of the Zea mays plant. Delaying flowering time usually results in reduced yield. In addition, linkage cargo affecting yield potential, particularly silage yield potential, may be distal and/or proximal to the Helminthosporium resistance locus at 8.06 bp in Zea mays. Flanking regions closely linked to this resistance locus may carry known linked cargo, but these regions may be limited by the interval located between the alleles of the SYN14136 marker and the MA0021 marker, and/or the interval located between the alleles of the MA0022 marker and the SYN4196 marker (Fig. 6). Thus, the plant of the present invention may be a plant of the Zea mays species endogenously containing a nucleic acid molecule of the present invention, wherein the flanking regions in the genome do not contain the interval derived from A619HT2 or A619HT3 located between the alleles of the SYN14136 marker and the MA0021 marker, or an interval derived from A619HT2 or A619HT3 located between the alleles of the MA0022 marker and the SYN4196 marker. In a preferred embodiment of the present invention, the plant is a plant of the species Zea mays endogenously containing a nucleic acid molecule of the present invention, wherein the flanking regions in the genome do not contain an interval derived from A619HT2 or A619HT3 located between the alleles of the marker SYN14136 and the marker PZE108077560, or an interval derived from A619HT2 or A619HT3 located between the alleles of marker PZE108093423 and marker MA0021, or an interval derived from A619HT2 or A619HT3 located between alleles of marker MA0022 and marker SYN4196.

- 10 047146

table 2

Primer sequences of KASP markers and their transfer to donor alleles (X allele and Y allele: describe biallelic SNP values)

SNP marker AGPvO2 marker position [base pairs] Primer alleles X(5'-3') [SEQ ID NO] Primer alleles Y(5'-3') [SEQ ID NO] Regular primer (5'-3') [SEQ ID NO] Donor alleles A619HT2 (SNP) SYN14136 131681497 25 26 27 A PZE-108077560 133189880 28 29 thirty A PZE-108093423 150279048 31 32 33 A MA0021 151907173 34 35 36 G MA0022 152046529 37 38 39 A SYN4196 161766769 40 41 42 WITH

For example, removal of concatenated cargo can be accomplished by genetic recombination during the crossing process between two maize plants, with one parent maize plant carrying the HT2 resistance locus. In addition to the use of traditional selection methods, to obtain genetic recombination, which results in the replacement of at least one of the donor intervals using the above linked cargo with genomic sequences of the recurrent parent, which, in a preferred embodiment of the present invention, do not contain unwanted genes , modern biotechnology offers the skilled person a variety of tools that can enable precision genetic engineering. Examples of known tools are meganucleases (Silva et al. 2011), homing endonucleases (Chevalier 2002), zinc finger nucleases, TALE nucleases (WO 2010/079430; WO 2011/072246) or CRISPR systems (Guy et al. 2013 ). These are artificial nuclease fusion proteins that are capable of cleaving double-stranded nucleic acid molecules, such as plant DNA, and thus producing double-strand breaks at desired positions in the genome. By using the cell's own machinery to repair induced double-strand breaks, homologous recombination or nonhomologous end joining can occur, which can lead to the removal of intervals of linked donor-carrying cargo. Suitable target sequences in the genome for domain nuclease recognition can be taken, for example, from SNP marker sequence information (Table 2). However, one skilled in the art will also be able to identify other sequences, in a preferred embodiment of the present invention, within certain flanking regions described above that are suitable as target sequences for nuclease recognition domains.

In another aspect, a genetically edited or transgenic plant is provided comprising a nucleic acid molecule of the present invention, a vector of the present invention, or an expression cassette of the present invention. The nucleic acid molecule may be a transgenic or a modified/edited endogenous gene. A promoter may be operably linked to a nucleic acid molecule or nucleotide sequence for expression.

Also provided is a part or seed of a plant of the present invention, containing a nucleic acid molecule of the present invention, a vector of the present invention, or an expression cassette of the present invention, wherein the seed contains a nucleic acid molecule of the present invention, a vector of the present invention, an expression cassette of the present invention invention. The nucleic acid molecule may be a transgenic or a modified/edited endogenous gene.

According to another aspect, a method is provided for identifying or selecting a plant, in a preferred embodiment of the present invention, a plant of the species Zea mays, having increased resistance to a plant disease caused by a pathogenic fungus, in a preferred embodiment of the present invention, Helminthosporium turcicum, in a more preferred embodiment of the present invention - Helminthosporium turcicum races 0, 1 and/or N,

- 11 047146 or part, cell or seed thereof, comprising the following steps: (a) determining in a plant or part thereof, cell or seed (or in a sample of a plant or part thereof, cell or seed) the presence of a nucleic acid molecule of the present invention as described above, or a nucleotide sequence selected from the group consisting of: (i) a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80% -th, 85%, 90%, 92%, 94%, 96%, 98%, 99% or 99.5% identity with the nucleotide sequence presented in SEQ ID NO: 1 or SEQ ID NO: 2, in a preferred embodiment of the present invention, the entire length of, (ii) a nucleotide sequence encoding an amino acid sequence having at least 60%, 65%, 70% new, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, 99% or 99.5% - identity with the amino acid sequence presented in SEQ ID NO: 3, in the preferred embodiment of the present invention - along the entire length, (iii) nucleotide sequence having at least 85%, 90%, 92% 94%, 96%, 98%, 99%, or 99.5% identity to nucleotide positions 1-920 of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO : 2 (exon 1; SEQ ID NO: 6), in a preferred embodiment of the present invention - along the entire length, and/or having at least 60%, 65%, 70%, 75%, 80% new, 85%, 90%, 92%, 94%, 96%, 98%, 99% or 99.5% identity with nucleotide positions 23252-23288 the nucleotide sequence presented in SEQ ID NO: 1 (exon 2; SEQ ID NO: 7), or with nucleotide positions 921-957 of the nucleotide sequence presented in SEQ ID NO: 2 (exon 2; SEQ ID NO: 7), in in a preferred embodiment of the present invention - along the entire length, and/or having at least 98%, 98.5%, 99%, 99.5%, 23586-24632%, 85% 90%, 92%, 94%, 96%, 98%, 99%, or 99.5% identity to nucleotide positions 23252-23288 of the nucleotide sequence set forth in SEQ ID NO: 1 (exon 3; SEQ ID NO: 8), or with nucleotide positions 958-2004 of the nucleotide sequence presented in SEQ ID NO: 2 (exon 3; SEQ ID NO: 8), in a preferred embodiment of the present invention, the entire length of, (iv) a nucleotide sequence encoding an amino acid sequence having at least 75%, 80%, 85%, 90% 92%, 94%, 96%, 98%, 99% or 99.5% identity with positions 1-306 of the amino acid sequence shown in SEQ ID NO: 3, in a preferred embodiment of the present invention - along the entire length, and/or having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, 99%, or 99.5% identity to positions 307-319 of the amino acid sequence shown in SEQ ID NO: 3, in a preferred embodiment of the present invention - along the entire length, and/or having at least 98%, 98.5%, 99% or 99.5% identity with amino acid positions 320-668 the sequence presented in SEQ ID NO: 3, in a preferred embodiment of the present invention - along its entire length, wherein the nucleic acid molecule according to any one of paragraphs (i) to (iv) encodes a polypeptide capable of conferring or increasing resistance to a plant disease, caused by a pathogenic fungus, in a preferred embodiment of the present invention - Helminthosporium turcicum, in a more preferred embodiment of the present invention - Helminthosporium turcicum race 0, 1 and/or N, in a plant, in a preferred embodiment of the present invention - in a plant of the species Zea mays, in which a polypeptide is expressed, and wherein, in a preferred embodiment of the present invention, the nucleic acid molecule of any one of clauses (i) to (iv) encodes a polypeptide that is or functionally belongs to the receptor-like kinase 1 (WAK RLK1) family , associated with the wall; or determining in a plant, or a part, cell or seed thereof (or in a sample of a plant, or a part, cell or seed thereof) the presence of a polypeptide encoded by a nucleic acid molecule of the present invention as described above, or a nucleotide sequence according to any of paragraphs (i )-(iv), and (b) identifying or selecting a plant in which, or in a part, cell or seed of which, a nucleic acid molecule as defined in paragraph (a) is present as having resistance or increased resistance to the plant disease caused by the pathogen fungus, in a preferred embodiment of the present invention - Helminthosporium turcicum, in a more preferred embodiment of the present invention - Helminthosporium turcicum race 0, 1 and/or N.

The method may include the additional step of obtaining a sample of nucleic acid from a plant or part thereof, a cell, or a seed and determining the sequence in the sample. For example, the step of obtaining a nucleic acid sample from a plant or part thereof, or seed may be carried out prior to determination step (a).

Determination of the nucleotide sequence can be carried out by hybridization using an oligonucleotide probe. Hybridization conditions can be adjusted accordingly depending on factors such as the Tm value of the probe used and the CG content of the target DNA. Known hybridization methods are described, for example, in Sambrook et al., 2001.

- 12 047146

In an alternative embodiment of the present invention, detection of the gene can be accomplished by a DNA amplification method, such as PCR, using appropriate primers. When the determination is carried out by PCR, the PCR conditions can be appropriately selected depending on factors such as the Tm value of the primer used and the length of the amplified region to be determined. Determination can be accomplished by amplifying the target by PCR and confirming the presence or absence of the PCR-amplified product. The method for confirming the presence or absence of an amplification product is not particularly limited. For example, the amplification product can be confirmed by subjecting the nucleic acid amplification reaction mixture to agarose gel electrophoresis; then, by staining the gel with an appropriate nucleic acid staining reagent such as ethidium bromide, SYBER Green I, etc.; and by determining the presence or absence of streaks resulting from irradiation with ultraviolet rays. The bands can be determined by visual observation, or they can be determined by using, for example, a fluorescence image analyzer or the like.

Also proposed is a method or method for identifying a plant, in a preferred embodiment of the present invention, a plant of the Zea mays species, having increased resistance to a plant disease caused by a pathogenic fungus, Helminthosporium turcicum, in a more preferred embodiment of the present invention, Helminthosporium turcicum race 0, 1 and/ or N, or a portion having increased resistance to the plant disease caused by Helminthosporium turcicum, in a preferred embodiment of the present invention is Helminthosporium turcicum race 0, 1 and/or N, and it contains a nucleic acid molecule of the present invention endogenously, a cell or a seed thereof , a method or method comprising identifying in plant alleles at least two markers, wherein at least one of said markers is located on or within a chromosomal interval between SYN14136 and a nucleic acid molecule of the present invention, and at least At least one of these markers is located on or within the chromosomal interval between the nucleic acid molecule of the present invention and SYN4196. In a preferred embodiment of the present invention, a method or method comprising identifying at least two markers in plant alleles, wherein at least one of said markers is located on or within a chromosomal interval between PZE108093423 and the nucleic acid molecule of the present the invention, and at least one of these markers is located on or within the chromosomal interval between the nucleic acid molecule of the present invention and SYN4196.

In another aspect, a method is provided for identifying a resistance gene allele that confers or increases resistance to a plant disease caused by a pathogenic fungus, in a preferred embodiment of the present invention - Helminthosporium turcicum, in a more preferred embodiment of the present invention - Helminthosporium turcicum race 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, in Zea mays and in a preferred embodiment of the present invention encoding a polypeptide that is or functionally belongs to the family of receptor-like kinase 1 (WAK RLK1) associated with a wall, the method comprising the following steps: (a) performing a sequence comparison using (i) at least one coding nucleotide sequence isolated from a Zea mays genotype, wherein the nucleotide sequence, in a preferred embodiment of the present invention , corresponds to the 8.05 bp resistance locus or the 8.06 bp resistance locus, and (ii), as a reference sequence, the nucleotide sequence of the present invention, in a preferred embodiment of the present invention the nucleotide sequence set forth in SEQ ID NO : 1 or SEQ ID NO: 2, or a nucleotide sequence encoding the amino acid sequence presented in SEQ ID NO: 3, or a part thereof, or a consensus sequence obtained from a set of at least two nucleotide sequences, wherein one nucleotide the sequence is a nucleotide sequence of the present invention, in a preferred embodiment of the present invention, the nucleotide sequence presented in SEQ ID NO: 1 or SEQ ID NO: 2, or a nucleotide sequence encoding the amino acid sequence presented in SEQ ID NO: 3, or parts thereof, and wherein each nucleotide sequence of the set of at least two nucleotide sequences encodes a polypeptide capable of conferring or increasing resistance to a plant disease caused by a pathogenic fungus, in a preferred embodiment of the present invention - Helminthosporium turcicum, in a more preferred embodiment implementation of the present invention - Helminthosporium turcicum race 0, 1 or N, in Zea mays, in which the polypeptide is expressed, and, in a preferred embodiment of the present invention, corresponds to an 8.05 bp resistance locus or an 8.06 bp resistance locus, and, in a preferred embodiment of the present invention, encoding a polypeptide that is or functionally belongs to the wall-associated receptor-like kinase 1 (WAK RLK1) family, and (b) identifying

- 13 047146 allele if sequence comparison reveals (i) nucleotide-level sequence identity of at least 85%, 90%, 92%, 94%, 96%, 98 %, 99%, or 99.5% identity to nucleotide positions 1-920 of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, in a preferred embodiment of the present invention, throughout its entire length, and /or a component of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94% 96%, 98%, 99% or 99.5% identity with nucleotide positions 23252-23288 of the nucleotide sequence presented in SEQ ID NO: 1, or with nucleotide positions 921-957 of the nucleotide sequence, presented in SEQ ID NO: 2, in the preferred embodiment of the present invention - along the entire length, and/or comprising at least 98%, 98.5%, 99% or 99.5% - exact identity with nucleotide positions 23586-24632 of the nucleotide sequence presented in SEQ ID NO: 1, or with nucleotide positions 958-2004 of the nucleotide sequence presented in SEQ ID NO: 2, in the preferred embodiment of the present invention - along the entire length, and/ or (ii) sequence identity at the encoded amino acid level of at least 75%, 80%, 85%, 90%, 92%, 94%, 96% , 98%, 99% or 99.5% identity with positions 1-306 of the amino acid sequence presented in SEQ ID NO: 3, in a preferred embodiment of the present invention - along the entire length, and/or component, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96% -, 98%, 99% or 99.5% identity with positions 307-319 of the amino acid sequence presented in SEQ ID NO: 3, in a preferred embodiment of the present invention - along the entire length, and/or component at least 98%, 98.5%, 99%, or 99.5% identity to positions 320-668 of the amino acid sequence set forth in SEQ ID NO: 3, in a preferred embodiment of the present inventions - along the entire length.

In a preferred embodiment of the present invention, the consensus sequence is derived from a set of at least two nucleotide sequences, wherein one nucleotide sequence is a nucleotide sequence of the present invention, in a preferred embodiment of the present invention, the nucleotide sequence set forth in SEQ ID NO : 1 or SEQ ID NO: 2, or a nucleotide sequence encoding the amino acid sequence presented in SEQ ID NO: 3, or a part thereof, and wherein another nucleotide sequence is selected from the group consisting of a nucleotide sequence presented in SEQ ID NO: 9, or a part thereof, the nucleotide sequence presented in SEQ ID NO: 11, or a part thereof, the nucleotide sequence presented in SEQ ID NO: 13, or a part thereof, the nucleotide sequence presented in SEQ ID NO: 23, or a part thereof, a nucleotide sequence encoding the amino acid sequence presented in SEQ ID NO: 10, or a part thereof, a nucleotide sequence encoding the amino acid sequence presented in SEQ ID NO: 12, or a part thereof, a nucleotide sequence encoding the amino acid sequence presented in SEQ ID NO: 14, or a part thereof, or a nucleotide sequence encoding the amino acid sequence presented in SEQ ID NO: 24 or a part thereof.

The at least one coding nucleotide sequence isolated from the Zea mays genotype of step (a)(i) may be obtained from a plant sequence database or from a gene bank in which samples of different Zea mays genotypes are stored. Plant sequence databases or gene banks are known in the art.

The method for identifying a (new) allele of a resistance gene (or a potential new allele of a resistance gene) conferring resistance or increased resistance to a plant disease caused by a pathogenic fungus may further comprise, for example, as step (c), the step of determining the level of resistance of the plant to the pathogenic fungus. fungus caused by the identified allele.

Also provided is a nucleic acid molecule containing or consisting of a nucleotide sequence encoding an allele of a resistance gene (or a potential new allele) identified by the identification method described above. Said allele may be used in a method for producing a plant, in a preferred embodiment of the present invention, a plant of the Zea mays species, or in a method for imparting/increasing resistance to a plant disease caused by a pathogenic fungus, such as Helminthosporium turcicum, in a preferred embodiment of the present invention, Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N. In one embodiment of the present invention, the method of the present invention for producing a plant or the method of the present invention for imparting/increasing resistance to a plant disease further comprises, in step (a), introducing at least one nucleic acid molecule containing or consisting of a nucleotide sequence, encoding a new resistance gene in at least one plant cell.

In another aspect, a method of imparting or increasing resistance to a plant disease caused by a pathogenic fungus is provided, in a preferred embodiment of the present invention.

- 14 047146 bretenia - Helminthosporium turcicum, in a more preferred embodiment of the present invention - Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, in a plant, in a preferred embodiment of the present invention - in a plant of the species Zea mays, comprising the following steps: (a) introducing into at least one cell of the plant a nucleic acid molecule of the present invention, including, for example, a nucleic acid molecule containing or consisting of the nucleotide sequence of an identified allele, an expression cassette of the present invention or a vector of the present invention, (b) regenerating a plant from at least one cell, and (c) causing expression of a nucleic acid molecule in the plant.

In a preferred embodiment of the method of conferring or increasing resistance to a plant disease caused by a pathogenic fungus, step (a) results in the modification of an endogenous nucleic acid molecule conferring sensitivity to a plant disease caused by a pathogenic fungus, in a preferred embodiment of the present invention - Helminthosporium turcicum, in in a more preferred embodiment of the present invention, Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, in a preferred embodiment of the present invention, corresponding to a resistance locus of 8.05 base pairs or an 8.06 base pair resistance locus on chromosome 8 of Zea mays, and, in a preferred embodiment of the present invention, encoding a polypeptide that is or functionally belongs to the wall-associated receptor-like kinase 1 (WAK RLK1) family, wherein , the modification converts an endogenous nucleic acid molecule into a nucleic acid molecule of the present invention conferring resistance to a plant disease caused by a pathogenic fungus, in a preferred embodiment of the present invention Helminthosporium turcicum, in a more preferred embodiment of the present invention Helminthosporium turcicum race 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, provided it is expressed.

In another preferred embodiment of a method of conferring or increasing resistance to a plant disease caused by a pathogenic fungus, step (a) results in the modification of an endogenous nucleic acid molecule conferring sensitivity to a plant disease caused by a pathogenic fungus, in a preferred embodiment of the present invention Helminthosporium turcicum, in in a more preferred embodiment of the present invention, Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, in a preferred embodiment of the present invention, corresponding to a resistance locus of 8.05 base pairs or an 8.06 base pair resistance locus on chromosome 8 of Zea mays, and, in a preferred embodiment of the present invention, encoding a polypeptide that is or functionally belongs to the wall-associated receptor-like kinase 1 (WAK RLK1) family, wherein, the modification converts an endogenous nucleic acid molecule into a nucleic acid molecule of the present invention conferring increased resistance to a plant disease caused by a pathogenic fungus, in a preferred embodiment of the present invention - Helminthosporium turcicum, in a more preferred embodiment of the present invention - Helminthosporium turcicum race 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, provided it is expressed.

Also proposed is a method for increasing resistance to a plant disease caused by a pathogenic fungus, in a preferred embodiment of the present invention - Helminthosporium turcicum, in a more preferred embodiment of the present invention - Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, in a plant of the present invention, comprising the steps of reducing the level of expression of a nucleic acid molecule of the present invention, including, for example, a nucleic acid molecule containing or consisting of the nucleotide sequence of an identified allele, expression cassette of the present invention, or vector of the present invention, in a plant or in at least one cell of a plant, in a preferred embodiment of the present invention, compared to the level of endogenous gene expression in a resistant wild-type plant. Such a resistant wild-type plant is, for example, selected from the lines A619HT2, B37HT2 or B73HT2. The reduction may be transient or long-term, in a preferred embodiment of the present invention as a preventative measure if infection by a pathogenic fungus is expected, for example due to special environmental conditions that influence the spread of the pathogenic fungus. Various methodologies for reducing gene expression in a plant are well known to those skilled in the art. Long-term reduction of expression levels can be achieved, for example, by modulating the promoter or other regulatory elements through random or targeted mutagenesis, or by stable integration of an expression cassette that allows the expression of double-stranded RNA, single-stranded antisense RNA or hairpin DNA that are capable of suppressing, like interfering RNA, m RNA encoded by the nucleic acid molecule of the present invention. Such an inhibitory RNA molecule, in the preferred embodiment of the present invention in the form of si RNA molecules, can also be used to transiently reduce the level of expression if it is applied to plants as a spray and actively or passively

- 15 047146 is taken up by plant cells, in a preferred embodiment of the present invention by leaf cells.

In another aspect, a method is provided for producing a plant (or part, cell or seed thereof), in a preferred embodiment of the present invention, a plant of the species Zea mays having (increased) resistance to a plant disease caused by a pathogenic fungus, in a preferred embodiment of the present invention - Helminthosporium turcicum, in a more preferred embodiment of the present invention - Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, or a part, cell or seed thereof, comprising the following steps: (a) introducing into a plant or at least one cell of a plant a nucleic acid molecule of the present invention, including, for example, a nucleic acid molecule containing or consisting of the nucleotide sequence of an identified allele, expression cassette of the present invention, or vector of the present invention , (b) optionally regenerating the plant from at least one cell; and (c) causing expression of a nucleic acid molecule in the plant or part thereof.

In a preferred embodiment of the method of the present invention for producing a plant (or part, cell or seed thereof) of the species Zea mays, step (a) results in the modification of an endogenous nucleic acid molecule conferring sensitivity to a plant disease caused by a pathogenic fungus, in a preferred embodiment of the present invention - Helminthosporium turcicum, in a more preferred embodiment of the present invention - Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, in a preferred embodiment of the present invention, corresponding to the resistance locus an 8.05 base pair or 8.06 base pair resistance locus on chromosome 8 of Zea mays, and, in a preferred embodiment of the present invention, encoding a polypeptide that is or functionally belongs to the receptor-like kinase 1 (WAK RLK1) family, associated with the wall, wherein the modification converts an endogenous nucleic acid molecule into a nucleic acid molecule of the present invention conferring resistance to a plant disease caused by a pathogenic fungus, in a preferred embodiment of the present invention Helminthosporium turcicum, in a more preferred embodiment of the present invention Helminthosporium turcicum race 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, as long as it is expressed.

In another preferred embodiment of the method of the present invention for producing a plant (or part, cell or seed thereof) of the Zea mays species, step (a) results in modification of an endogenous nucleic acid molecule conferring resistance to a plant disease caused by a pathogenic fungus, in a preferred embodiment of the present invention - Helminthosporium turcicum, in a more preferred embodiment of the present invention - Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, in a preferred embodiment of the present invention, corresponding to the locus 8.05 bp resistance or 8.06 bp resistance locus on chromosome 8 of Zea mays, and, in a preferred embodiment of the present invention, encoding a polypeptide that is or functionally belongs to the receptor-like kinase 1 (WAK RLK1) family associated with the wall, wherein the modification converts an endogenous nucleic acid molecule into a nucleic acid molecule of the present invention conferring increased resistance to a plant disease caused by a pathogenic fungus, in a preferred embodiment of the present invention - Helminthosporium turcicum, in a more preferred embodiment of the present invention - Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, subject to its expression.

In a preferred embodiment of the present invention, the method consists of obtaining a plant (or part, cell or seed thereof) having increased resistance to a plant disease caused by a pathogenic fungus such as Helminthosporium turcicum, preferably Helminthosporium turcicum race 0, 1, 2, 3 , N, 12, 23, 2N, 12N, 23N and/or 123N. Accordingly, the method may comprise the additional step (d) of selecting a plant having increased resistance compared to a reference plant. In a preferred embodiment of the present invention, the reference plant is a plant of the same species, in a more preferred embodiment of the present invention, the reference plant is a wild type plant of the same species or a plant that is isogenic to the plant selected in step (d) , expected for a nucleic acid molecule of the present invention that was introduced in step (a) or modified in step (a) as described above.

In a preferred embodiment of the present invention, in the methods of the present invention, a nucleic acid molecule of the present invention is expressed in a plant or portion thereof in an amount and/or for a period sufficient to enhance or confer resistance to a plant disease caused by a pathogenic fungus in the plant.

In another aspect, a plant produced by the methods of the present invention, or its progeny, fruit or seed, is provided. In a preferred embodiment of the present invention,

- 16 047146 a plant or its progeny, fruit or seed contains a nucleic acid molecule of the present invention or a vector of the present invention, or a protein of the present invention, and/or a cell of the present invention.

In another aspect, the use of at least one nucleic acid molecule of the present invention is provided, including, for example, a nucleic acid molecule containing or consisting of the nucleotide sequence of an identified novel allele of a resistance gene, to produce a plant having increased resistance to a plant disease, caused by a pathogenic fungus, in a preferred embodiment of the present invention Helminthosporium turcicum, in a more preferred embodiment of the present invention Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N. The resulting plant may be a genetically edited or transgenic plant.

Also proposed is a method for combating infection by a pathogenic fungus, in a preferred embodiment of the present invention - Helminthosporium turcicum, in a more preferred embodiment of the present invention - Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and /or 123N, comprising the following steps: (a) growing plants of the present invention in agricultural and horticultural areas and (b) causing expression of a nucleic acid molecule of the present invention, including, for example, a nucleic acid molecule containing or consisting of the nucleotide sequence of the identified allele , an expression cassette of the present invention or a vector of the present invention, in plants.

Another aspect of the present invention is an oligonucleotide having a length of at least 15, 16, 17, 18, 19 or 20, in a preferred embodiment of the present invention at least 21, 22, 23, 24 or 25, in a more preferred embodiment in an embodiment of the present invention, at least 30, 35, 40, 45, 50, 100, 200, 300 or 500 nucleotides, wherein the oligonucleotide is capable of hybridizing or annealing to (i) a nucleic acid molecule of the present invention, (ii) a a nucleic acid containing the nucleotide sequence represented in SEQ ID NO: 4 or SEQ ID NO: 5, or (iii) a nucleic acid molecule complementary to (i) or (ii). In a preferred embodiment of the present invention, the oligonucleotide contains a nucleotide sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98% 98.5%, 99%, 99.5% or 100% identity with the nucleotide sequence of the nucleic acid molecule from points (i), (ii) or (iii), with which the oligonucleotide is capable hybridize or anneal.

Also provided is a pair of oligonucleotides or a set containing said oligonucleotides, wherein the oligonucleotides are suitable for annealing as a forward primer and a reverse primer to a region in the plant genome, in a preferred embodiment of the present invention, in the Zea mays genome, which exhibits cosegregation, in a preferred embodiment implementation of the present invention - ideal cosegregation with the nucleic acid molecule of the present invention. In a preferred embodiment of the present invention, the pair contains one or two oligonucleotides of the present invention. In a preferred embodiment of the present invention, the kit contains one or at least two oligonucleotides of the present invention.

In another aspect, the use of at least one oligonucleotide as a molecular marker or part thereof is provided in a method for identifying or selecting a plant, in a preferred embodiment of the present invention, a plant of the Zea mays species having increased resistance to a plant disease caused by a pathogenic fungus, in a preferred embodiment in an embodiment of the present invention - Helminthosporium turcicum, in a more preferred embodiment of the present invention - Helminthosporium turcicum race 0, 1 and/or N, or a part, cell or seed thereof, or in a method for identifying an allele of a resistance gene that confers or increases resistance to a plant disease caused by a pathogenic fungus, in a preferred embodiment of the present invention - Helminthosporium turcicum, in a more preferred embodiment of the present invention - Helminthosporium turcicum race 0, 1 and/or N, in Zea mays, wherein the molecular marker is capable of detecting at least one single nucleotide polymorphism, to diagnose a deletion or insertion for a nucleic acid molecule of the present invention and/or contains an oligonucleotide as described above and/or is a pair of oligonucleotides or a set as described above. Such a single nucleotide polymorphism, deletion, or insertion can be directly obtained from the sequence alignment shown in FIG. 2. In a preferred embodiment of the present invention, at least one single nucleotide polymorphism, deletion or insertion results in an exchange, deletion or insertion of at least one amino acid. In table 2 shows representative amino acid exchanges, deletions or insertions from a comparison of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 3 with the RLK 1 polypeptide (SEQ ID NO: 10) conferring the Pepitilla resistance phenotype disclosed in WO 2015/032494.

In another aspect, a method is provided for determining the presence or absence of a nucleic acid molecule

- 17 047146 voyl acid according to the present invention, including, for example, a nucleic acid molecule containing or consisting of the nucleotide sequence of an identified allele, in a plant, in a preferred embodiment of the present invention in a plant of the species Zea mays, comprising the following steps: (a) isolation DNA from at least one plant cell and (b) using a molecular marker to determine the presence or absence of a nucleic acid molecule of the present invention, wherein the molecular marker is capable of detecting at least one single nucleotide polymorphism, deletion or insertion diagnosis for the molecule nucleic acid of the present invention and/or contains an oligonucleotide as described above, and/or is a pair of oligonucleotides or a set as described above.

One aspect provides the use of at least one oligonucleotide or at least two oligonucleotides as a molecular marker or part thereof in a method or method for identifying a plant, as described above, in a method or method for eliminating cohesive cargo closely linked to a nucleic acid molecule according to the present invention, to determine the presence or absence of an interval derived from A619HT2 or A619HT3 located between the alleles of the SYN14136 marker and the MA0021 marker, or an interval derived from A619HT2 or A619HT3 located between the alleles of the MA0022 marker and the SYN4196 marker. In a preferred embodiment of the present invention, at least one oligonucleotide is used as a molecular marker or part thereof in a method or method for eliminating concatenated cargo closely linked to a nucleic acid molecule of the present invention to determine the presence or absence of an A619HT2-derived interval or A619HT3, located between the alleles of the SYN14136 marker and the PZE108077560 marker, the interval obtained from A619HT2 or A619HT3, located between the alleles of the PZE108093423 marker and the MA0021 marker, and/or the interval obtained from A619HT2 or A619HT3, located between alleles of the MA0022 marker and the SYN4196 marker.

In another aspect, there is provided a method for determining the presence of a nucleic acid molecule of the present invention, including, for example, a nucleic acid molecule containing or consisting of the nucleotide sequence of an identified allele, in a plant, in a preferred embodiment of the present invention, in a plant of the species Zea mays, comprising the following steps : (a) isolating DNA from at least one plant cell; and (b) using a molecular marker to determine the presence or absence of a nucleic acid molecule of the present invention, wherein the molecular marker is capable of detecting at least one single nucleotide polymorphism, making a diagnosis deletions or insertions for a nucleic acid molecule of the present invention and/or contains an oligonucleotide as described above and/or is an oligonucleotide pair or set as described above. Such a single nucleotide polymorphism, deletion, or insertion can be directly obtained from the sequence alignment shown in FIG. 2. In a preferred embodiment of the present invention, at least one single nucleotide polymorphism, deletion or insertion results in an exchange, deletion or insertion of at least one amino acid. In table 3 shows representative amino acid exchanges, deletions or insertions from a comparison of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 3 with the RLK 1 polypeptide conferring the Pepitilla resistance phenotype (SEQ ID NO: 10) disclosed in WO 2015/032494.

Table 3. Representative amino acid exchanges, deletions or insertions from a comparison of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 3 with the RLK 1 polypeptide (SEQ ID NO: 10) conferring the Pepitilla resistance phenotype disclosed in WO 2015/032494 ( see also Fig. 3).

- 18 047146

Table 3

Position HTN NT2 Effect exon 5 Q L strong Exon 1 7 n R weak Exon 1 9 S R weak Exon 1 27 G A weak Exon 1 36 N S weak Exon 1 51 A V strong Exon 1 56 E Exon 1 75 I T strong Exon 1 95 E TO weak Exon 1 101 S R weak Exon 1 102 R T weak Exon 1 114 G D strong Exon 1 115 D N weak Exon 1 122 S Exon 1 123 Y Exon 1 127 Q Y weak Exon 1 128 Q N moderate Exon 1 135 R S moderate Exon 1 142 G E strong Exon 1 147 R N moderate Exon 1 157 L Exon 1 158 N Exon 1 162 A R moderate Exon 1 180 N D weak Exon 1 182 R L strong Exon 1 187 D G strong Exon 1 188 Y N weak Exon 1 196 N S weak Exon 1 199 T A moderate Exon 1

- 19 047146

204 R G strong Exon 1 210 T R weak Exon 1 211 G E moderate Exon 1 216 Q N weak Exon 1 217 E A strong Exon 1 223 L S strong Exon 1 235 R S weak Exon 1 236 D E weak Exon 1 239 Q Exon 1 246 F L weak Exon 1 249 T G moderate Exon 1 250 R Exon 1 256 V L moderate Exon 1 261 F I strong Exon 1 266 N TO weak Exon 1 275 R Q weak Exon 1 278 G E moderate Exon 1 284 W R strong Exon 1 297 L F weak Exon 1 303 V A strong Exon 1 305 S N weak Exon 1 314 T Exon 2 315 TO Exon 2 316 To R weak Exon 2 318 To E weak Exon 2 319 E A strong Exon 2 320 G A weak Exon 2 321 R S weak Exon 2 345 G WITH strong Exon 3 352 E To weak Exon 3 368 S Exon 3 566 I T strong Exon 3

Definitions

The term resistance or resistance, when applied to a pathogen, should be understood as the ability of a plant, plant tissue or plant cell to resist the damaging effects of the pathogen and includes the manifestation of effects from delaying the development of the disease to completely suppressing the development of the disease. Resistance may be complete or partial and may be specific or nonspecific to the pathogen race. Conferred resistance may be newly inherited resistance or an increase in already existing partial resistance.

Stability can be quantified by methods known in the art. For example, resistance to Helminthosporium turcicum can be quantified by determining classification scores using phenotyping experiments according to the scheme shown in Table 4 below. For example, a corn plant resistant to Helminthosporium turcicum within the meaning of the present invention exhibits increased resistance to H. turcicum by at least 1 classification point, in a preferred embodiment of the present invention by at least 2 classification points or by at least 3 classification points, and in the most preferred embodiment of the present invention at least 4 classification points. In a preferred embodiment of the present invention, a corn plant in accordance with the present invention exhibits resistance to at least one race of Helminthosporium turcicum that does not conform to the known race specificity known in the art. In a particularly preferred embodiment of the present invention, the corn plant of the present invention is resistant to all known races of Helminthosporium turcicum, that is, the conferred resistance is not race specific and may be particularly advantageous in generating broad resistance to Helminthosporium turcicum.

Table 4. Scheme of classification points in phenotyping experiments in pilot tests carried out in various locations with natural and artificial inoculation of H. turcicum (based on materials of the German Corn Committee (Deutsche Maiskomitee, DMK); variety AG from 02/27/02; (DMK, J . Rath; District Office of Freiburg H.J. Imgraben).

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Table 4

Classification score Phenotype 1 Plants do not show disease symptoms, 0% 2 Beginning of infection, small spots (less than 2 cm) are visible at first. Less than 5% of the leaf surface is affected. 3 Some spots developed at the leaf stage. 5-10% of the leaf surface is affected. 4 10-20% of the leaf surface is affected. Spots are clearly visible at several stages of the leaf. 5 20-40% of the leaf surface is affected. The spots begin to coalesce. 6 40-60% of the leaf surface is affected. Systemic infection is noticeable on the leaves. 7 60-80% of the leaf surface is affected. About half of the leaves are spoiled or withered due to fungal infection. 8 80-90% of the leaf surface is affected. More than half of the leaves are spoiled or withered due to infection by the fungus. 9 90-100% of the leaf surface is affected. The plants are almost completely dry.

The term hybridize or hybridization should be understood as a procedure in which a single-stranded nucleic acid molecule agglomerates with a nucleic acid strand that is maximally complementary, that is, forms a complementary base pair with it. Examples of standard hybridization methods were described in 2001 by Sambrook et al. In a preferred embodiment of the present invention, it is intended to be at least 60%, in a more preferred embodiment of the present invention, at least 65%, 70% , 75%, 80% or 85%, in a particularly preferred embodiment of the present invention - 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the bases of the nucleic acid molecule acids undergo base pairing with the nucleic acid strand that is maximally complementary. The possibility of such agglomeration depends on the severity of the hybridization conditions. The term stringency refers to the hybridization conditions. High rigidity is said when base pairing is more difficult, and low rigidity is said when base pairing is easier. The stringency of the hybridization conditions depends, for example, on the salt concentration or ionic strength and temperature. Generally, hardness can be increased by increasing the temperature and/or decreasing the salt content. The term stringent hybridization conditions should be understood as those conditions under which hybridization occurs predominantly only between homologous nucleic acid molecules. The term hybridization conditions in this aspect refers not only to the actual conditions prevailing during the actual agglomeration of the nucleic acids, but also to the conditions prevailing during subsequent washing steps. Examples of extremely stringent hybridization conditions are those in which only those nucleic acid molecules that have at least 90% or at least 95% sequence identity are hybridized. Such extremely stringent hybridization conditions are, for example: hybridization in 4x SSC at 65°C followed by multiple washes in 0.1x SSC at 65°C for approximately 1 hour. As used herein, the term extremely stringent hybridization conditions can also mean: hybridization at 68°C in 0.25 M sodium phosphate, pH 7.2, 7% SDS, 1 mM EDTA and 1% BSA for 16 hours, followed by two washes 2x SSC and 0.1% SDS at 68°C. In a preferred embodiment of the present invention, hybridization occurs under harsh conditions. Less stringent hybridization conditions are, for example: hybridization in 4x SSC at 37°C, followed by multiple washes in 1x SSC at room temperature.

The present invention covers nucleic acid molecules containing or consisting of a nucleotide sequence encoding a protein, wherein said protein is derived from the amino acid sequence encoded by the nucleotide sequence presented in SEQ ID NO: 1 or SEQ ID NO: 2, or from the amino acid sequence presented in SEQ ID NO: 3, by substitution, deletion and/or addition of at least one amino acid. As used herein, the term at least one amino acid includes, for example, 1 to 50, 1 to 40, 1

- 21 047146 to 30 or from 1 to 20, in a preferred embodiment of the present invention from 1 to 10, in a more preferred embodiment of the present invention from 1 to 7, in an even more preferred embodiment of the present invention from 1 to 5 and in a particularly preferred embodiment of the present invention, 1, 2 or 3 amino acids.

Functionally linked means linked into a common nucleic acid molecule in such a way that the linked elements are arranged and oriented towards each other in such a way that transcription of the nucleic acid molecule can occur. DNA that is operably linked to a promoter is regulated by that promoter at the transcriptional level.

The term introduction as used herein includes stable integration through transformation, including Agrobacterium-mediated transformation, transfection, microinjection, biolistic bombardment, insertion using gene editing technology such as CRISPR systems (e.g. CRISPR/Cas, in particular CRISPR/Cas9 or CRISPR/Cpf1), CRISPR/CasX or CRISPR/CasY), TALEN, zinc finger nucleases or meganucleases, homologous recombination optionally through one of the above gene editing technologies, including, in a preferred embodiment of the present invention, a repair template , modification of an endogenous gene using random or targeted mutagenesis such as TILLING or the aforementioned gene editing technology and the like. The term introduction may or may not cover introgression using traditional selection.

The term transgenic or transgene means that the corresponding gene is an exogenous gene that has been introduced into the plant. An exogenous gene may be obtained from a species different from the plant species into which it was introduced. In an alternative embodiment of the present invention, the corresponding gene may be a gene already present in the plant species into which it is introduced, such that at least one additional copy of the gene is present as a result of the introduction of the transgene. The term transgenic plant is a plant into which at least one polynucleotide has been integrated into its genome, in a preferred embodiment of the present invention a heterologous polynucleotide. In a preferred embodiment of the present invention, the polynucleotide has been integrated in a stable manner, which means that the integrated polynucleotide is stably maintained in the plant, expressed and can also be stably transmitted to progeny. The term heterologous means that the introduced polynucleotide originates, for example, from the same cell or organism having a different genetic background of the same species or a different species, or it is homologous to a prokaryotic or eukaryotic host cell, but is then placed in a different genetic environment and thus thus, it is different from any possible corresponding naturally occurring polynucleotide. A heterologous polynucleotide may be present in addition to the corresponding endogenous gene.

Plant organs are leaves, plant stems, infructescences, roots, vegetative buds, meristems, embryos, anthers, fertilized eggs or fruits. The term plant part can mean a fusion of several organs, such as a flower or a seed, or a part of an organ, such as a transverse segment from a stem. Examples of plant tissues are callus tissue, storage tissue, meristematic tissue, embryogenic tissue, leaf tissue, bud tissue, root tissue, plant tumor tissue or reproductive tissue. The term plant cells refers, for example, to isolated plant cells having a cell wall, or their aggregates, or protoplasts.

The term genetically modified means that an endogenous gene has been modified, for example through random mutagenesis, TILLING or gene editing technology. For example, according to the present invention, an endogenous plant gene can be modified to confer resistance (or increased resistance) to a plant disease caused by a pathogenic fungus. Genetic modification can be achieved, for example, using methods of random or targeted mutagenesis (such as gene editing) or homologous recombination (optionally using gene editing tools) or combinations thereof.

As used herein, the term modification means that the genetic sequence has changed by at least one nucleotide. This may occur by substitution of at least one nucleotide and/or deletion of at least one nucleotide, and/or insertion of at least one nucleotide until a complete change of at least one nucleotide is achieved from the nucleotide sequence before modification, which allows the modification to be identified, for example, using methods such as sequencing or PCR analysis and the like, of which one skilled in the art is well aware.

The term allele refers to one or at least two nucleotide sequences at a specific locus in the genome. The first allele is on one chromosome, the second allele is on the sister chromosome in the same position. If these two alleles are different, then they are heterozygous, and if they are the same, then they are homozygous. Different alleles of a gene (gene alleles) differ in at least one SNP. Different alleles of a resistance gene can either confer resistance, perhaps varying levels of resistance, to a pathogenic fungus, that is, cause different types of plant phenotypes in response to infection by a pathogenic fungus, or construct a variant of the gene that is not capable of conferring resistance. that is, the resulting plant phenotype is sensitive to the pathogenic fungus.

According to the present invention, the term regulatory sequence means a nucleotide sequence that affects the specificity and/or level of expression, for example, to the extent that the regulatory sequence confers a certain tissue specificity. A regulatory sequence of this type may be located upstream of the transcription initiation point of a minimal promoter, but also downstream of it, for example, in a transcribed but untranslated leader sequence or in an intron.

A molecular marker or marker is a nucleotide sequence that is used as a starting or targeting point. A marker to recognize a recombination event must be suitable for monitoring differences or polymorphisms in a plant population. For markers, these differences exist at the DNA level, and they are, for example, differences in polynucleotide sequences, such as, for example, SSRs (simple sequence repeats), RFLPs (restriction fragment length polymorphisms), FLPs (fragment length polymorphisms) or SNPs (single nucleotide polymorphisms). Markers can be derived from genomic or expressed nucleic acids, such as spliced RNA, cDNA or EST, and they can be based on nucleic acids that are used as probes or primer pairs and, as such, are suitable for amplifying a sequence fragment using PCR based methods. Markers that relate to genetic polymorphisms between parts of a population can be determined using known methods from the prior art (Introduction to Genetic Analysis. 7th edition, Griffith, Miller, Suzuki et al., 2000). These include, for example: DNA sequencing, PCR-based sequence-specific amplification, quantitative RFLP analysis, quantitative KASP analysis, quantitative analysis of polynucleotide polymorphisms using allele-specific hybridization (ASH), determination of SSRs, SNPs or AFLPs. Methods for detecting EST (expressed sequence tags) and RAPD (random amplified polymorphic DNA) are also known. Depending on the context, the term marker as used herein may also refer to a specific chromosomal position in the genome of a species in which a specific marker (eg, SNP) may be identified.

The terms distal and proximal describe the position of a chromosomal interval or genetic segment relative to a specific starting point (eg, a particular polynucleotide, another chromosomal interval, or gene) on the entire chromosome; distal means that the interval or segment is located on the side of the origin far from the centromere of the chromosome, and proximal means that the interval or segment is located on the side of the origin close to the centromere of the chromosome.

The term closely linked means two loci, two intervals, two genetic segments (for example, a resistance gene and flanking regions) or two markers (marker loci) that are less than 15 cm, less than 12 cm, less than 10 cm, less than 8 cm apart. less than 7 cm, less than 6 cm, less than 5 cm, less than 4 cm, less than 3 cm, less than 2 cm, less than 1 cm, less than 0.5 cm, less than 0.2 cm, less than 0.1 cm apart, installed using the IBM2 nearest neighbors genetic map 4, which is publicly available on the Maize GDB website, or that are less than 50 megabp apart, less than 40 megabp, less than 30 megabp, less 25 mega base pairs, less than 20 mega base pairs, less than 15 mega base pairs, or less than 10 mega base pairs.

The term interval or chromosomal interval means a continuous linear segment on genomic DNA that is present on a single chromosome in a plant or on a fragment of a chromosome, and which is usually defined by two markers that represent the endpoints of the interval on the distal and proximal sides. In this regard, markers defining the ends of an interval may themselves also be part of the interval. In addition, two different intervals may overlap. As used herein, an interval is characterized by the expression between marker A and marker B. The end marker of the interval may also be located in a specific marker region on one side of the interval. The marker region is then defined by providing two flanking markers and is a chromosomal segment on which more markers can be located in addition to the flanking markers. Flanking markers define the endpoints of the marker area and are themselves still part of the marker area. If both end markers of an interval are markers located in different marker regions on either side of the interval, then, as used herein, the interval is characterized by the expression between a marker in marker region X, which is flanked by markers C and D, and a marker in marker region Y, which is flanked by markers E and F. The marker region may span up to 500,000 base pairs and, in a preferred embodiment of the present invention, its size may be from 100,000 to 400,000 base pairs, or, in a particularly preferred embodiment of the present invention, it may be from 140,000 to 315,000 base pairs.

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The term introgression, as used in connection with the present invention, means the transfer of at least one desired gene allele at a genetic locus of a genetic background to another. For example, introgression of a desired gene allele at a particular locus can be transferred to an offspring by a sexual cross between two parents of the same species. In an alternative embodiment of the present invention, for example, transfer of a gene allele may also occur by recombination between two donor genomes in a fused protoplast, wherein at least one donor protoplast carries the desired gene allele in its genome. In each case, offspring that then contain the desired gene allele can be backcrossed back to the line that contains the preferred genetic background and can be selected for the desired gene allele. The result is the fixation of the desired gene allele in the selected genetic background.

The term locus means the position on the chromosome where at least one gene is identified that causes or influences an agronomic trait. In particular, the term locus as used herein means the HT2 resistance locus, which confers resistance to the pathogen Helminthosporium turcicum or at least the Helminthosporium turcicum race.

The term allele refers to one or at least two nucleotide sequences at a specific locus in the genome. The first allele is on one chromosome, the second allele is on the second chromosome in the same position. If these two alleles are different, then they are heterozygous, and if they are the same, then they are homozygous. Different alleles of a gene (gene alleles) differ in at least one SNP. Depending on the context of the present description, an allele also means a single SNP that, for example, allows a distinction between a resistance donor and a recurrent parent.

Examples

The examples below, including experiments performed and results achieved; they are for illustrative purposes only and are not intended to limit the present invention.

1. Cloning and functional validation of the HT2 and HT3 resistance genes.

a) QTL (quantitative trait locus) mapping and development of recombinants.

The A619HT2 donor line was crossed and backcrossed to the RP1 line to create a nearly isogenic line (NIL, RP1HT2A) with a major fragment of the original donor A619HT2 on chromosome 8 and very few other small donor regions. This NIL line RP1HT2A was crossed with its recurrent parent RP1 to create the F2 population. The same was done for RP2 x RP2HT3A (the original donor in this case was A619HT3). Recurrent parents RP1 and RP2 were sensitive to NCLB (scores 7-9; see Table 4), while donor lines A619HT2 and A619HT3 are resistant (scores 1-3). The NIL lines RP1HT2A and RP2HT3A have scores of 2-3 and 1-2 respectively.

F2 populations were planted in the field as 720 individuals at a site located in Pocking, Germany. QTL mapping resulted in a peak (LOD value for population with RP2HT3A = 168.77; LOD value for population with RP1HT2A = 44.02) in both populations on chromosome 8 (1.1 cm frame of 3.5 megabp). No other significant peaks were identified. Recombinant plants (~2000 in total) were developed over several generations to F11 status. QTL mapping in F2 and further fine mapping with recombinants narrowed the chromosomal region to a physical interval of 490 kb (genetic interval = 0.2 cm). This interval includes the RLK1 gene.

b) Molecular analysis of the target region.

From the RP1HT2A and RP2HT3A lines, blank BAC (bacterial artificial chromosome) libraries were developed and screened with 7 target locus probes. For the RLK1 candidate gene region, a contiguous BAC contig could be developed for both donor lines. Sequence analysis showed that the donor lines A619HT2 and A619HT3 are identical for the target region (of 1 megabase pairs).

The cDNA sequence of the PLK1 candidate gene shows 97 polymorphisms (at the DNA level; includes SNPs and Insertion/Deletions) and 61 amino acid changes (at the protein level) between lines containing HTM1 from WO 2015/032494 A2 and the HT2 allele. Of the 97 polymorphisms, only 14 single nucleotide polymorphisms (SNPs) result in silent amino acid exchanges that likely do not affect resistance gene activity. All other polymorphisms significantly change the protein structure through substitution, addition, or deletion. In table Figure 3 shows 15 amino acid exchanges that are predicted to have major effects on protein structure. Among these, multiple genotype-specific additional amino acids have been identified. In comparison with the cDNA sequences of the RLK1 gene indicated in SEQ ID NO: 11 and 13 from the donor line disclosed in WO 2011/163590 A1, the donor lines A619HT2 and A619HT3 differ by 31 nucleotides and 12 amino acids.

c) Functional validation of the HT2 allele of the RLK 1 gene.

(i) Functional validation using EMS mutagenesis.

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An EMS mutagenesis population from RP2HT3A was developed. exon regions 1 and 3 of RLK1 were screened and 3 positive mutants carrying an amino acid change were identified (SEQ ID NOs: 16, 18, 20, 46, 48, 50, 52, 54, 56 and 58). In the cDNA of the RLK gene Mutant WVE16-92125-001, G at position 1625 is replaced by A (see also SEQ ID NO: 15), leading to an amino acid exchange from Glycine to Aspartic acid at position 542 (see also SEQ ID NO: 16 ); in the cDNA of the RLK gene Mutant WVE16-92149-012, C at position 95 is replaced by T (see also SEQ ID NO: 17), leading to an amino acid exchange from Proline to Leucine at position 32 (see also SEQ ID NO: 18) ; in the cDNA of the RLK gene Mutant WVE16-92168-005 G at position 115 is replaced by A (see also SEQ ID NO: 19), leading to an amino acid exchange from Alanine to Threonine at position 39 (see also SEQ ID NO: 20) ; in the cDNA of the RLK gene Mutant WVE16-92168-024_WVE17-68687-013 G at position 73 is replaced by A (see also SEQ ID NO: 45), leading to an amino acid exchange from Alanine to Threonine at position 25 (see also SEQ ID NO: 46); in the cDNA of the RLK gene Mutant WVE17-68655-008, C at position 301 is replaced by T (see also SEQ ID NO: 47), leading to an amino acid exchange from Proline to Serine at position 101 (see also SEQ ID NO: 48) ; in the cDNA of the RLK gene Mutant WVE17-68625-014 G at position 715 is replaced by A (see also SEQ ID NO: 49), leading to an amino acid exchange from Valine to Isoleucine at position 239 (see also SEQ ID NO: 50) ; in the cDNA of the RLK gene Mutant WVE17-68611-006, T at position 862 is replaced by A (see also SEQ ID NO: 51), leading to an amino acid exchange from Phenylalanine to Isoleucine at position 288 (see also SEQ ID NO: 52) ; in the cDNA of the RLK gene Mutant WVE17-68696-002 A at position 929 is replaced by G (see also SEQ ID NO: 53), leading to an amino acid exchange from Lysine to Arginine at position 310 (see also SEQ ID NO: 54) ; in the cDNA of the RLK gene Mutant WVE17-68656-011, C at position 1289 is replaced by T (see also SEQ ID NO: 55), leading to an amino acid exchange from Threonine to Isoleucine at position 430 (see also SEQ ID NO: 56) ; in the cDNA of the RLK gene Mutant WVE17-68630-001 G at position 1826 is replaced by A (see also SEQ ID NO: 57), leading to an amino acid exchange from Cysteine to Tyrosine at position 609 (see also SEQ ID NO: 58) . After these mutants selfed, they were evaluated in the field and greenhouse.

d) Gene expression analysis.

Analysis of PLK1 gene expression in RP1, RP1HT2A, RP2 and RP2HT3A in uninfected and infected leaf material showed similar gene expression in NIL lines RP1HT2A and RP2HT3A. Gene expression is suppressed in infected leaf material. This response to infection could also be shown for the HTN1 allele of the RLK 1 gene.

2. Allelic sequence of the RLK 1 gene and identification of the relevant region for the resistance response.

In order to identify the relevant region for the resistance response to the pathogen Helminthosporium turcicum, the following analysis is carried out: Bioinformatic analysis of resequencing of the RLK1 gene in various donor lines and recurrent parents identifies the amino acid region relevant for the resistance response. Therefore, genomic sequence primer pairs for the RLK1 gene were designed to span the exonic regions. exon 1 could be covered only partially, and exons 2 and 3 completely. The developed amplicons were amplified and sequenced in 96 genotypes using the PACBio sequencing method. Sequences were assembled into consensus sequences for each genotype. Multiple consensus sequences were obtained for some genotypes. The consensus sequence with the highest number of sequencing reads was selected to assemble all 96 genotypes. G aplotypes were determined according to the exonic region/parts. For exon 1, 12 different haplotypes were identified, for exon 2-7 different haplotypes, and for exon 3-9 different haplotypes. Genetic distance was calculated using Lasergene MegAlign software (DNASTAR, Inc.). The different haplotypes were collected at the DNA and Protein sequence level (see Table 5). As a result, exon 1 and 2 regions appear to be highly variable within the gene and contain WAK-associated domains. This part of the protein is located in the intercellular space and could interact with fungal proteins. Variations in these two exons represent candidate base pairs of interest for this interaction. Based on this, it is possible to identify exon 1 and 2 haplotypes in new alleles of the RLK1 gene that are capable of conferring or increasing resistance to at least the pathogen Helminthosporium turcicum.

In addition, phenotyping of various donor lines, near-isogenic lines and recurrent parents and haplotype analysis of the RLK1 gene locus are carried out, as well as analysis of RLK1 gene expression in various donor lines, near-isogenic lines and recurrent parents in combination with phenotypic analysis to identify the relevant region for the reaction resistance and, finally, to identify new allelic variants of the resistance gene. Evaluating all of the data sets described should narrow down the relevant area for the resistance response.

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Claims (11)

Table 5 Genetic distances [%] between different haplotypes based on exons Homology at the DNA level Homology at the protein level Final sequence: > 60% Final sequence: > 60% Exon 1: >85% Exon 1: >75% Exon 2: >60% Exon 2: >60% Exon 3: >98% Exon 3: >98% 3. Resistance reaction to other pathogens. A set of genotypes containing different alleles of the RLK 1 gene were inoculated with other plant pathogens such as Southern corn leaf blight (Bipolaris maydis), Corn rust (Puccinia sorghi) and Diplodia macrospora (Stenocarpella macrospora). A common feature of these pathogens is that the infection is caused by very similar fungal biology and the fact that they enter the host through leaf tissue. The first experiment with southern corn leaf blight (Bipolaris maydis) also indicates a resistance response of the HT2 and HTN alleles of the RLK1 gene. 4. Introduction of resistance to NCLB caused by Helminthosporium turcicum into a susceptible genotype through Agrobacterium-mediated transformation. Three different constructs with the HT2 cDNA sequence (SEQ ID NO: 2) under the influence of promoters with different activities were transformed into the sensitive maize genotype A188: Construct A with the HT2 cDNA sequence (SEQ ID NO: 2), a native promoter region of ~1980 base pairs (SEQ ID NO: 4) and terminator region (SEQ ID NO: 5) (vector p7U, see Fig. 1), construct B with HT2 cDNA sequence (SEQ ID NO: 2), Oryza sativa actin promoter (SEQ ID NO : 43) and terminator region (SEQ ID NO: 5), and construct C with HT2 cDNA sequence (SEQ ID NO: 2), Brachypodium distachyon EF1 promoter (SEQ ID NO: 44) and terminator region (SEQ ID NO: 5) . To obtain, for example, the p7U-nativeHT2_CDS_2 vector, an expression cassette containing the HT2 gene under the control of the native promoter and terminator was transformed into a binary vector containing a herbicide gene (for example: resistance to BASTA, resistance to glyphosate or resistance to ALS inhibitors) for subsequent transformation in Agrobacterium tumefaciens for Agrobacterium-mediated plant transformation into maize (Zea mays) genotype A188. T0 plants stably transformed with the various constructs were propagated, and greenhouse-grown offspring were tested for NCLB resistance. Transgenic plants with all the different constructs showed increased resistance to NCLB, however, to varying degrees depending on the site of integration and promoter strength. 5. Introduction of resistance to NCLB caused by Helminthosporium turcicum into a susceptible genotype through gene editing. The PLK1 gene allele in the susceptible maize genotype A188 was identified, sequenced, and cDNA predicted (SEQ ID NO: 21). Comparison of the cDNA of this A188 allele with the cDNA of the HT2 gene shows that the sequences show 99% sequence identity (Fig. 4). However, at position 1458-1459 of the A188 allele there is an AC insertion, 2 base pairs long, which causes an early stop codon after Cysteine at position 513 (SEQ ID NO: 22). By eliminating this 2-bp insertion using gene editing based on the TALEN or CRISPR systems, the stability of the HT2 gene could be restored in the A188 genotype. In fig. Figure 5 shows the alignment between the RLK1 gene protein obtained from the modified RLK1 gene allele of genotype A188 (SEQ ID NO: 24), and the HT2 gene allele with a high level of amino acid sequence identity, cDNA of the modified RLK1 gene allele from genotype A188 is shown in SEQ ID NO: 23. CLAIM 1. A nucleic acid molecule containing a nucleotide sequence selected from the group consisting of (a) a nucleotide sequence presented in SEQ ID NO: 1 or SEQ ID NO: 2; (b) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 3; (c) a nucleotide sequence having at least 99% identity with the nucleotide sequence presented in SEQ ID NO: 1 or SEQ ID NO: 2 throughout its length; and (d) a nucleotide sequence encoding an amino acid sequence having at least 99% identity with the amino acid sequence presented in SEQ ID NO: 3 throughout its length; - 26 047146 characterized in that the nucleic acid molecule encodes a polypeptide capable of imparting or increasing resistance to a plant disease caused by Helminthosporium turcicum race 0, 1 and N, and shows a sensitive response to infection by Helminthosporium turcicum races 2 and/or 3 in the plant in which the polypeptide is expressed, compared to a reference plant, which is a plant of the same species. 2. A method for identifying an allele of a resistance gene, characterized in that the allele confers increased resistance to a plant disease caused by Helminthosporium turcicum races 0, 1 and N in Zea mays compared to a reference plant, which is a plant of the same species, containing the following steps: (a) performing sequence comparisons using (i) at least one coding nucleotide sequence derived from a Zea mays genotype, wherein the nucleotide sequence corresponds to an 8.05 bp resistance locus or an 8.06 bp resistance locus, and, (ii) as a reference sequence, the nucleotide sequence of the nucleic acid molecule according to claim 1, or a part thereof, or a consensus sequence obtained from a set of at least two nucleotide sequences, wherein one nucleotide sequence is the nucleotide sequence of the nucleic acid molecule according to claim 1 1 or a part thereof, and the additional nucleotide sequence is selected from the group consisting of the nucleotide sequence specified in SEQ ID NO: 9, or a part thereof, the nucleotide sequence specified in SEQ ID NO: 11, or a part thereof, the nucleotide sequence specified. in SEQ ID NO: 13, or part thereof, the nucleotide sequence specified in SEQ ID NO: 23, or part thereof, the nucleotide sequence encoding the amino acid sequence specified in SEQ ID NO: 10, or part thereof, the nucleotide sequence encoding the amino acid the sequence specified in SEQ ID NO: 12, or a part thereof, a nucleotide sequence encoding the amino acid sequence specified in SEQ ID NO: 14, or a part thereof, or a nucleotide sequence encoding the amino acid sequence specified in SEQ ID NO: 24, or parts thereof, wherein each nucleotide sequence of the set of at least two nucleotide sequences encodes a polypeptide capable of conferring or increasing resistance to a plant disease caused by Helminthosporium turcicum in Zea mays in which the polypeptide is expressed, and corresponds to an 8.05 bp resistance locus bases or resistance locus of 8.06 base pairs; wherein the portion of the nucleic acid molecule according to claim 1 is a nucleotide sequence having at least 98% identity to positions 2358624632 of the nucleotide sequence specified in SEQ ID NO: 1 (exon 3; SEQ ID NO: 8), or positions 958-2004 the nucleotide sequence specified in SEQ ID NO: 2, or is a nucleotide sequence encoding an amino acid sequence having at least 98% identity to positions 320-668 of the amino acid sequence specified in SEQ ID NO: 3; and (b) allele identification if sequence comparison reveals i) sequence identity at the nucleotide level constituting at least 85% identity with nucleotide positions 1-920 of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and constituting at least 60% identity with nucleotide positions 23252-23288 of the nucleotide sequence presented in SEQ ID NO: 1, or with nucleotide positions 921-957 of the nucleotide sequence presented in SEQ ID NO: 2, and/or having at least 98% identity with the nucleotide positions 23586-24632 of the nucleotide sequence presented in SEQ ID NO: 1, or with nucleotide positions 958-2004 of the nucleotide sequence presented in SEQ ID NO: 2, and/or ii) sequence identity at the amino acid level of at least 75% - at least 60% identical to positions 1-306 of the amino acid sequence shown in SEQ ID NO: 3 and/or at least 60% identical to positions 307-319 of the amino acid sequence shown in SEQ ID NO: 3 and at least at least 98% identity with positions 320-668 of the amino acid sequence presented in SEQ ID NO: 3. 3. A vector or expression cassette containing a nucleic acid molecule according to claim 1. 4. Vector or expression cassette according to claim 3, characterized in that the nucleic acid molecule is functionally linked to a promoter that ensures expression of the nucleotide sequence in the plant cell. 5. A polypeptide encoded by a nucleic acid molecule according to claim 1. 6. A plant or part thereof containing a nucleic acid molecule according to claim 1, a vector or expression cassette according to claims 3, 4 or a polypeptide according to claim 5. 7. A plant according to claim 6, characterized in that the plant is a genetically modified plant or a transgenic plant. 8. A plant or part thereof according to claim 6, characterized in that the plant or part thereof endogenously contains - 27 047146 containing the nucleic acid molecule according to claim 1 and the flanking regions of the genome, closely linked to the nucleic acid molecule, does not contain the interval carrying the linked cargo, obtained from the donor lines A619HT2 or A619HT3, located between the alleles of the SYN14136 marker and the MA0021 marker, or carrying the linked cargo interval obtained from donor lines A619HT2 or A619HT3, located between the alleles of the MA0022 marker and the SYN4196 marker; where the SYN14136 marker defines a single nucleotide polymorphism at position 131681497 relative to the reference gene B73 AGPv02, detected using primers SEQ ID NO: 25-27, the MA0021 marker defines a single nucleotide polymorphism at position 151907173 relative to the reference gene B73 AGPv02, detected using primers SEQ ID NO: 34-36, the MA0022 marker defines a single nucleotide polymorphism at position 152046529 in relation to the B73 AGPv02 reference gene, detected using primers SEQ ID NO: 37-39, and the SYN4196 marker defines a single nucleotide polymorphism at position 161766769 in relation to the B73 AGPv02 reference gene, detected using primers SEQ ID NO: 40-42. 9. A plant seed according to claim 6, containing a nucleic acid molecule according to claim 1, a vector or expression cassette according to claims 3, 4 or a polypeptide according to claim 5. 10. A method for identifying or selecting a plant having increased resistance to a plant disease caused by Helminthosporium turcicum races 0, 1 and N, compared to a reference plant, which is a plant of the same species, or a part, cell or seed thereof, comprising the following steps : (a) determining in a plant or part thereof, cell or seed the presence of a nucleic acid molecule according to claim 1 or a nucleic acid molecule containing or consisting of a nucleotide sequence selected from the group consisting of i) a nucleotide sequence having at least 99% identity over its entire length with the nucleotide sequence presented in SEQ ID NO: 1 or SEQ ID NO: 2; ii) a nucleotide sequence encoding an amino acid sequence having at least 99% identity over its entire length with the amino acid sequence presented in SEQ ID NO: 3, wherein the nucleic acid molecule encodes a polypeptide capable of conferring or increasing resistance to a plant disease, caused by a pathogenic fungus in a plant in which the polypeptide is expressed; iii) a nucleotide sequence having at least 85% identity with nucleotide positions 1-920 of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 (exon 1; SEQ ID NO: 6), or having at least 60% identity with nucleotide positions 23252-23288 of the nucleotide sequence presented in SEQ ID NO: 1 (exon 2; SEQ ID NO: 7), or nucleotide positions 921-957 of the nucleotide sequence presented in SEQ ID NO: 2 (exon 2; SEQ ID NO: 7), and having at least 98% identity with nucleotide positions 23586-24632 of the nucleotide sequence shown in SEQ ID NO: 1 (exon 3; SEQ ID NO: 8), or positions nucleotides 958-2004 of the nucleotide sequence shown in SEQ ID NO: 2; iv) a nucleotide sequence encoding an amino acid sequence having at least 75% identity with positions 1-306 of the amino acid sequence presented in SEQ ID NO: 3, and/or having at least 60% identity with positions 307- 319 amino acid sequence presented in SEQ ID NO: 3, and having at least 98% identity with positions 320-668 of the amino acid sequence presented in SEQ ID NO: 3; characterized in that the nucleic acid molecule according to any one of claims (1) to (1y) encodes a polypeptide capable of imparting or increasing resistance to a plant disease caused by Helminthosporium turcicum in a plant in which the polypeptide is expressed; (b) identification or selection of a plant in which, or in a part, cell or seed of which, a nucleic acid molecule defined in paragraph (a) is present as having resistance or increased resistance to the plant disease caused by a pathogenic fungus. 11. A method for increasing resistance to a plant disease caused by Helminthosporium turcicum races 0, 1 and N, in comparison with a reference plant, which is a plant of the same species, containing the following steps: (a) introducing into at least one plant cell a nucleic acid molecule according to claim 1, or a vector or expression cassette according to claim 3, (b) regenerating the plant from at least one cell, (c) causing expression of the nucleic acid molecule at the plant. -