Patent Application
20210071194
The present invention relates to a nucleic acid molecule encoding a polypeptide conferring resistance to a plant against a fungal pathogen, such as Helminthosporium turcicum. The present invention further relates to a plant (or part thereof) comprising the nucleic acid molecule, and methods involving the nucleic acid molecule.
In maize (Zea mays L.), there are many fungal pathogens which cause leaf diseases. The fungus which can cause by far the most damage under tropical and also under temperate climatic conditions, such as those in large parts of Europe and North America as well as in Africa and India, is known as Helminthosporium turcicum or synonymously as 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 in epidemic proportions during wet years, attacking vulnerable maize varieties and causing a great deal of damage and considerable losses of yield of 30% and more over wide areas. Since the 1970s, then, natural resistance in genetic material has been sought. Currently, quantitative and qualitative resistances, even if incomplete, are known. While the oligo- or polygenically inherited quantitative resistance appears incomplete and non-specific as regards race in the phenotype and is influenced by additional and partially dominant genes, qualitative resistance seems to be typically race-specific and can be inherited through individual, mostly dominant genes at loci like HT1, HT2, HT3, Htm1 or HTN1 (Lipps et al., 1997, “Interaction of Ht and partial resistance to Exserohilum turcicum in maize.” Plant Disease 81: 277-282; Welz & Geiger, 2000, “Genes for resistance to northern corn leaf blight in diverse maize populations.” Plant Breeding 119: 1-14). Backcrosses in many frequently used inbred maize lines such as W22, A619, B37 or B73 have successfully brought about introgression of the HT loci, where they exhibit a partial dominance and expression as a function of the respective genetic background (Welz, 1998, “Genetics and epidemiology of the pathosystem Zea mays/Setosphaeria turcica” Habilitationsschrift, Institut für Pflanzenzüchtung, Saatgutforschung und Populations-genetik, Universität Hohenheim).
Despite this complex genetic architecture of NCLB resistance in maize, until now, principally, the use of the HT1 gene located on the long arm of chromosome 2 together with a partial quantitative resistance has been sufficient to control helminthosporiosis in maize (Welz, 1998). The basis for this is that globally, races 0 and 1 of H. turcicum are most prevalent (approximately 55%) (Lipps et al., 1997), while other races such as 2N and 23N are only rare and even in geographically restricted areas (Welz, 1998). This race 0 is avirulent with regard to a maize plant with HT1 (see also Table 1), so that when provided with a suitable quantitative resistance, it exhibits a sufficient general resistance to NCLB. However, many studies have reported an increasing dissemination of the less common races (Jordan et al., 1983, “Occurrence of race 2 of Exserohilum turcicum on corn in the central and eastern United States.” Plant Disease 67: 1163-1165; Welz, 1998). The reasons for this are linked to the population dynamic of a pathogen which allows changes in pathogen virulence by new mutations in avirulence genes and new combinations of available virulence genes. This can lead to the occurrence of new, sometimes more aggressive pathogenic races. In Brazil, for example, the H. turcicum population already appears to be substantially more diverse with regard to the race composition than, for example, in North America. Already in the 1990s it has been reported that H. turcicum races had broken the resistance conferred by the HT1 gene. In addition, there is the instability of the resistance genes to certain environmental factors such as temperature and light intensity in some climate zones (Thakur et al., 1989, “Effects of temperature and light on virulence of Exserohilum turcicum on corn.” Phytopathology 1989, 79: 631-635). As a consequence, the use of novel HT resistance genes for the production of commercial maize plants in order to target a broader and more long-lasting resistance to H. turcicum in maize is growing in importance.
One source of monogenic HTN1 resistance is the Mexican landrace “Pepitilla” (Gevers, 1975, “A new major gene for resistance to Helminthosporium turcicum leaf blight of maize.” Plant Dis Rep 59: 296-300). HTN1 introgression lines exhibit a gene mapping on the long arm of chromosome 8. In contrast to the usual HT resistance genes, HTN1 confers resistance by delaying the onset of sporulation, and thus combats the development of lesions. As a result, fewer, smaller lesions as well as reduced sporulation zones are formed (Simcox & Bennetzen, 1993, “The use of molecular markers to study Setospaeria turcica resistance in maize.” Phytopathology 83: 1326-1330). Chlorotic-necrotic lesions such as those which occur with HT1, HT2 or HT3-conferred resistance, are not formed (Gevers, 1975). WO2015/032494 discloses the identification of the causative gene, RLK1, conferring the “Pepitilla” resistance phenotype on bin 8.06 in corn and describes molecular markers which are suitable to benefit from this resistance locus without close-linked, undesired linkage drag leading to a negative impact on the yield potential.
In WO2011/163590 the genotypes PH99N and PH26N have been discloses as alternative sources for NCLB resistance on chromosome 8 bin 5.
With the intention of identifying a resistance gene for NCLB from the maize hybrid DK888, in 2010, Chung et al. published a study for fine mapping the bin 8.06 resistance locus (Chung et al. 2010 “Characterization and fine-mapping of a resistance locus for northern leaf blight in maize bin 8.06” Theoretical and Applied Genetics 121(2): 205-227). Investigations on Helminthosporium race specificity initially made it clear that functionally, the resistance locus was closely linked with the HT2 and HTN1 genes. Genome annotations of a 0.46 Mb-sized chromosome fragment using B73 reference hinted at several putative open reading frames; however, the causative gene has not been identified and a functional verification was not described.
It is thus an objective of the present invention to identify and/or further characterize plant resistance genes encoding polypeptides conferring or increasing resistance against a fungal pathogen, such as Helminthosporium turcicum.
The present invention provides a nucleic acid molecule comprising or consisting of a nucleotide sequence having the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. Also provided is a nucleic acid molecule comprising or consisting of a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 3.
Further provided is a nucleic acid molecule comprising or consisting of a nucleotide sequence having at least 96% identity to the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 and a nucleic acid molecule comprising or consisting of a nucleotide sequence encoding an amino acid sequence having at least 92% identity to the amino acid sequence set forth in SEQ ID NO: 3.
Also provided is a nucleic acid molecule comprising or consisting of a nucleotide sequence hybridizing with the complementary strand of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 or hybridizing with the complementary strand of the nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 3 under stringent hybridization conditions.
Moreover, provided is a nucleic acid molecule comprising or consisting of a nucleotide sequence encoding a protein, said protein being derived from the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or from the amino acid sequence set forth in SEQ ID NO: 3 by way of substitution, deletion and/or addition of one or more amino acid(s).
The nucleic acid molecule according to the present invention encodes a polypeptide which is a wall associated receptor-like kinases 1 (WAK RLK1) or functionally belongs to the family of wall associated receptor-like kinases.
The nucleic acid molecule of the invention is encoding a polypeptide capable of conferring (or increasing) resistance to a plant disease caused by fungal pathogen, in particular by Helminthosporium turcicum, preferably by Helminthosporium turcicum races 0, 1 and/or N in a plant, preferably in a plant of the species Zea mays, in which the polypeptide is expressed.
In another aspect, provided is a vector or expression cassette comprising the nucleic acid molecule of the present invention. Preferably, in the vector or expression cassette, the nucleotide sequence of the invention is operably linked to a regulatory element allowing expression of the nucleotide sequence in a plant cell.
Further provided is a host cell comprising the nucleic acid molecule of the invention or the vector or expression cassette of the invention.
In another aspect, a polypeptide encoded by the nucleic acid molecule of the invention is provided. The polypeptide is a wall associated receptor-like kinases 1 (WAK RLK1) or functionally belongs to the family of wall associated receptor-like kinases 1. Preferably, the polypeptide is capable of conferring (or increasing) resistance to a plant disease caused by Helminthosporium turcicum, preferably by Helminthosporium turcicum races 0, 1 and/or N in a plant, preferably in a plant of the species Zea mays, in which the polypeptide is expressed.
Additionally, the polypeptide may not be capable of conferring or increasing resistance to a plant disease caused by Helminthosporium turcicum races 2 and/or 3 in a plant, preferably a plant of the species Zea mays, in which the polypeptide is expressed. The plant may show a susceptible response to infection with Helminthosporium turcicum races 2 and/or 3.
Furthermore, provided is a plant, preferably a plant of the species Zea mays, comprising the nucleic acid molecule of the invention transgenically (as transgene) or endogenously (as endogenous gene), the vector of the invention, the expression cassette of the invention, or the polypeptide of the invention. Preferably, the plant is resistant to a plant disease caused by Helminthosporium turcicum, in particular by Helminthosporium turcicum races 0, 1 and/or N. The plant may show a susceptible response to infection with Helminthosporium turcicum races 2 and/or 3. The plant may be a transgenic plant or a genetically edited plant. The plant may be a plant comprising the nucleic acid molecule of the invention endogenously, wherein the genomic flanking regions does not contain an A619HT2 or A619HT3 derived interval located between alleles of marker SYN14136 and marker MA0021 or an A619HT2 or A619HT3 derived interval located between alleles of marker MA0022 and marker SYN4196. A part of the plant of the invention, plant cell of the plant of the invention and seed of the plant of the invention is also provided, wherein the seed comprising the nucleic acid molecule of the invention transgenically (as transgene) or endogenously (as endogenous gene), the vector of the invention, the expression cassette of the invention, or the polypeptide of the invention.
According to another aspect, provided is a method or process of identifying or selecting a plant, preferably a plant of the species Zea mays, having increased resistance to a plant disease caused by fungal pathogen, preferably by Helminthosporium turcicum, more preferably by Helminthosporium turcicum races 0, 1 and/or N, or a part, a cell or a seed thereof, comprising the following steps: (a) detecting in the plant, or part, cell or seed thereof (or in a sample of the plant, or part, cell or seed thereof), the presence of the nucleic acid molecule of the present invention as described above or a nucleic acid molecule comprising or consisting of a nucleotide sequence selected from the group consisting of: (i) a nucleotide sequence having at least 60% identity to the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, (ii) a nucleotide sequence encoding an amino acid sequence having at least 60% identity to the amino acid sequence set forth in SEQ ID NO: 3, (iii) a nucleotide sequence having at least 85% 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) and/or having at least 60% identity to nucleotide positions 23252-23288 of the nucleotide sequence set forth in SEQ ID NO: 1 (Exon 2; SEQ ID NO: 7) or to nucleotide positions 921-957 of the nucleotide sequence set forth in SEQ ID NO: 2 (Exon 2; SEQ ID NO: 7) and/or having at least 98% identity to nucleotide positions 23586-24632 of the nucleotide sequence set forth in SEQ ID NO: 1 (Exon 3; SEQ ID NO: 8) or to nucleotide positions 958-2004 of the nucleotide sequence set forth 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 to the positions 1-306 of amino acid sequence set forth in SEQ ID NO: 3 and/or having at least 60% identity to positions 307-319 of amino acid sequence set forth in SEQ ID NO: 3 and/or having at least 98% identity to positions 320-668 of amino acid sequence set forth in SEQ ID NO: 3, wherein the nucleic acid molecule of any one of (i) to (iv) is encoding a polypeptide capable of conferring or increasing resistance to a plant disease caused by fungal pathogen, preferably by Helminthosporium turcicum, more preferably by Helminthosporium turcicum races 0, 1 and/or N, in a plant, preferably a plant of the species Zea mays, in which the polypeptide is expressed, and wherein preferably the nucleic acid molecule of any one of (i) to (iv) encodes a polypeptide which is or belongs functionally to the family of wall associated receptor-like kinases 1 (WAK RLK1); and (b) identifying or selecting the plant in which or in whose part, cell or seed the nucleic acid molecule as defined in (a) is present, as having a resistance or an increased resistance to a plant disease caused by a fungal pathogen, preferably by Helminthosporium turcicum, more preferably by Helminthosporium turcicum races 0, 1 and/or N.
Further provided is, a method or process of identifying a plant, preferably a plant of the species Zea mays, which is resistant to a plant disease caused by Helminthosporium turcicum, in particular by Helminthosporium turcicum races 0, 1 and/or N and comprises the nucleic acid molecule of the invention endogenously, the method or process comprising detecting in the plant alleles of at least two markers, wherein at least one of said markers is on or within the chromosomal interval between SYN14136 and the nucleic acid molecule of the invention, and at least one of said markers is on or within the chromosomal interval between the nucleic acid molecule of the invention and SYN4196.
In another aspect, provided is a plant identified or selected by the method or process of identifying or selecting according to the present invention, or progeny thereof.
In another aspect, provided is a method of identifying an allele of a resistance gene conferring or increasing resistance to a plant disease caused by a fungal pathogen, preferably by Helminthosporium turcicum, more preferably by Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, in Zea mays and preferably encoding a polypeptide which is or belongs functionally to the family of wall associated receptor-like kinases 1 (WAK RLK1), wherein the method comprises the following steps: (a) conducting sequence comparison using (i) at least one coding nucleotide sequence originating or derived from a Zea mays genotype, wherein the nucleotide sequence preferably maps to bin 8.05 resistance locus or to bin 8.06 resistance locus, and (ii) as reference sequence, a nucleotide sequence of the invention, preferably the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 or the nucleotide sequence encoding the amino acid sequence set forth 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 invention, preferably the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 or the nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 3, or a part 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 fungal pathogen, preferably by Helminthosporium turcicum, more preferably by Helminthosporium turcicum races 0, 1 and/or N, in Zea mays in which the polypeptide is expressed, and preferably maps to bin 8.05 resistance locus or to bin 8.06 resistance locus and preferably encoding a polypeptide which is or belongs functionally to the family of wall associated receptor-like kinases 1 (WAK RLK1); and (b) identifying the allele, if the sequence comparison reveals (i) a sequence identity on nucleotide level of at least 85% identity to nucleotide positions 1-920 of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 and/or of at least 60% identity to nucleotide positions 23252-23288 of the nucleotide sequence set forth in SEQ ID NO: 1 or to nucleotide positions 921-957 of the nucleotide sequence set forth in SEQ ID NO: 2 and/or of at least 98% identity to nucleotide positions 23586-24632 of the nucleotide sequence set forth in SEQ ID NO: 1 or to nucleotide positions 958-2004 of the nucleotide sequence set forth in SEQ ID NO: 2, and/or (ii) a sequence identity of encoded amino acid level of at least 75% identity to the positions 1-306 of amino acid sequence set forth in SEQ ID NO: 3 and/or of at least 60% identity to positions 307-319 of amino acid sequence set forth in SEQ ID NO: 3 and/or of at least 98% identity to positions 320-668 of amino acid sequence set forth in SEQ ID NO: 3.
Further provided is a nucleic acid molecule comprising or consisting of a nucleotide sequence of the allele of a resistance gene identified by the method of identifying an allele of a resistance gene.
In another aspect, provided is a method for conferring or increasing resistance to a plant disease caused by a fungal pathogen, preferably by Helminthosporium turcicum, more preferably by Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, in a plant, preferably a plant of the species Zea mays, comprising the following steps: (a) introducing into at least one cell of the plant the nucleic acid molecule of the present invention, including for example a nucleic acid molecule comprising or consisting of a nucleotide sequence of the identified allele, the expression cassette of the invention or the vector of the invention, (b) regenerating the plant from the at least one cell, and (c) causing expression of the nucleic acid molecule in the plant.
Further provided is a method for increasing resistance to a plant disease caused by a fungal pathogen, preferably by Helminthosporium turcicum, more preferably by Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, in a plant of the invention, comprising the step of reducing the level of expression of the nucleic acid molecule of the present invention, including for example a nucleic acid molecule comprising or consisting of a nucleotide sequence of the identified allele, the expression cassette of the invention or the vector of the invention, in the plant or at least one cell of the plant, preferably compared to the expression level of the endogenous gene in a resistant wild type plant.
In a further aspect, provided is a method for producing a plant, preferably a plant of the species Zea mays, having (increased) resistance to a plant disease caused by fungal pathogen, preferably by Helminthosporium turcicum, more preferably by Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, or a part, a cell or a seed thereof, comprising the following steps: (a) introducing into the plant or at least one cell of the plant the nucleic acid molecule of the present invention, including for example a nucleic acid molecule comprising or consisting of a nucleotide sequence of the identified allele, the expression cassette of the invention or the vector of the invention, (b) optionally, regenerating the plant from the at least one cell, and (c) causing expression of the nucleic acid molecule in the plant.
In another aspect, provided is a plant produced by the methods for producing a plant according to the present invention, or progeny thereof.
Further provided is a method for controlling infestation of a fungal pathogen, preferably Helminthosporium turcicum, more preferably Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, in a population of plants, preferably plants of the species Zea mays, comprising the following steps: (a) growing plants of the present invention on agricultural and horticultural fields, and (b) causing expression of the nucleic acid molecule of the present invention, including for example a nucleic acid molecule comprising or consisting of a nucleotide sequence of the identified allele, the expression cassette of the invention or the vector of the invention, in the plants.
Another aspect of the invention is an oligonucleotide having a length of at least 15, 16, 17, 18, 19 or 20, preferably at least 21, 22, 23, 24 or 25, more preferred at least 30, 35, 40, 45, 50, 100, 200, 300 or 500 nucleotides, wherein the oligonucleotide is able to hybridize or anneal to (i) the nucleic acid molecule of the invention, (ii) the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 5, or (iii) nucleic acid molecule complementary to (i) or (ii).
Further provided is a pair of oligonucleotides or a kit comprising said oligonucleotides, wherein the oligonucleotides are suitable to anneal as forward primer and reverse primer to a region in the plant genome, preferably the Zea mays genome, which shows a cosegregation, preferably a perfect cosegregation, with the nucleic acid molecule of the invention.
In a further aspect, provided is the use of at least one oligonucleotide as a molecular marker or part thereof in the method of identifying or selecting a plant, preferably a plant of the species Zea mays, having increased resistance to a plant disease caused by fungal pathogen, preferably by Helminthosporium turcicum, more preferably by Helminthosporium turcicum races 0, 1 and/or N, or a part, a cell or a seed thereof, or in the method of identifying an allele of a resistance gene conferring or increasing resistance to a plant disease caused by a fungal pathogen, preferably by Helminthosporium turcicum, more preferably by Helminthosporium turcicum races 0, 1 and/or N, in Zea mays, wherein the molecular marker is able to detect at least one single nucleotide polymorphism, deletion or insertion diagnostic for the nucleic acid molecule of the present invention and/or comprises the oligonucleotide as described above and/or is the pair of oligonucleotides or the kit as described above.
In one aspect, provided is the use of at least one or at least two oligonucleotide(s) as a molecular marker or part thereof in method or process of identifying a plant as described above or in a method or process for the elimination of linkage drag close linked to the nucleic acid molecule of the invention for detecting the presence or absence of an A619HT2 or A619HT3 derived interval located between alleles of marker SYN14136 and marker MA0021 or an A619HT2 or A619HT3 derived interval located between alleles of marker MA0022 and marker SYN4196.
In another aspect, provided is a method for detecting the presence or absence of the nucleic acid molecule of the invention, including for example a nucleic acid molecule comprising or consisting of a nucleotide sequence of the identified allele, in a plant, preferably a plant of the species Zea mays, comprising the following steps: (a) isolating DNA from at least one cell of the plant, and (b) using a molecular marker to detect the presence or absence of the nucleic acid molecule of the invention, wherein the molecular marker is able to detect at least one single nucleotide polymorphism, deletion or insertion diagnostic for the nucleic acid molecule of the present invention and/or comprises the oligonucleotide as described above and/or is the pair of oligonucleotides or the kit as described above.
The present invention is based on the identification of the HT2 gene which encodes a polypeptide conferring (or increasing) resistance to a plant against a fungal pathogen, such as Helminthosporium turcicum.
Accordingly, in one aspect of the present invention, provided is a nucleic acid molecule comprising or consisting of a nucleotide sequence encoding said HT2 gene. In one embodiment, the nucleic acid molecule of the invention is comprising or consisting of a nucleotide sequence having the nucleotide sequence set forth in SEQ ID NO: 1 (genomic DNA of HT2) or SEQ ID NO: 2 (cDNA of HT2). Also provided is a nucleic acid molecule comprising or consisting of a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 3 (HT2 protein).
Further provided is a nucleic acid molecule comprising or consisting of a nucleotide sequence having at least 96% identity to the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, preferably over the full length. Preferably, the nucleotide sequence having at least 97%, 98%, 99% or 99.5% identity to the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, preferably over the full length. Also provided is a nucleic acid molecule comprising or consisting of a nucleotide sequence encoding an amino acid sequence having at least 92% identity to the amino acid sequence set forth in SEQ ID NO: 2, preferably over the full length. Preferably, the nucleic acid molecule comprises 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 to the amino acid sequence set forth in SEQ ID NO: 3, preferably over the full length.
In another embodiment, provided is a nucleic acid molecule comprising or consisting of a nucleotide sequence hybridizing with the complementary strand of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 or the nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 3 under stringent hybridization conditions.
Moreover, provided is a nucleic acid molecule comprising or consisting of a nucleotide sequence encoding a protein derived from the protein encoded by the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or derived from the amino acid sequence set forth in SEQ ID NO: 3 by way of substitution, deletion and/or addition of one or more amino acid(s).
Deletion, substitution, or addition of amino acid(s) can be carried out by a technique known in the art. Mutagenesis in a nucleotide sequence can be caused by a known method such as the Kunkel method, the gapped duplex method, or a method similar to such a known method. For instance, mutagenesis can be caused with the use of a mutagenesis kit (e.g., Mutant-K or Mutant-G (product name, TAKARA Bio)) based on a site-directed mutagenesis method, an LA PCR in vitro Mutagenesis series kit (product name, TAKARA Bio), or the like. Alternatively, a mutagenesis method may be a method using a chemical mutagen represented by EMS (ethyl methanesulfonate), 5-bromouracil, 2-aminopurine, hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine, or a different 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 altered, the amino acid is preferably mutated for a different amino acid(s) that conserves 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 group-containing side chains (S, T, and Y), amino acids containing sulfur-containing side chains (C and M), amino acids containing carboxylic acid- and amide-containing 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 one-letter codes in parentheses). Amino acid substitutions within each group are called conservative substitutions. Conservative substitutions are preferred.
Preferably, the nucleic acid molecule of the invention is encoding a polypeptide capable of conferring (or increasing) resistance to a plant disease caused by a fungal pathogen in a plant in which the polypeptide is expressed.
Additionally, the nucleic acid molecule of the invention is encoding a polypeptide which may not be capable of conferring resistance to a plant disease caused by Helminthosporium turcicum races 2 and/or 3 in a plant, preferably a plant of the species Zea mays, in which the polypeptide is expressed. The plant may show a susceptible response to infection with Helminthosporium turcicum races 2 and/or 3.
A further example of the nucleic acid molecule of the invention may be a nucleic acid molecule comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 23, or a nucleic acid molecule comprising or consisting of a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 24.
In one embodiment, the fungal pathogen belongs to the division of Ascomycota or Basidiomycota. The fungal pathogen may belong to family Pleosporaceae, Pucciniaceae or Botryosphaeriaceae. Preferably, the fungal pathogen belongs to the genus of Setosphaeria, Bipolaris, Puccinia or Diplodia, more preferably is the species of Helminthosporium turcicum, Setosphaeria rostrata, Setosphaeria glycinea, Setosphaeria holmii, Setosphaeria khartoumensis, Setosphaeria minor, Setosphaeria monoceras, Setosphaeria pedicellata, Setosphaeria prolata, Bipolaris australis, Bipolaris brizae, Bipolaris buchloës, 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 Ieersiae, Bipolaris micropus, Bipolaris miyakei, Bipolaris multiformis, Bipolaris nicotiae, Bipolaris novae-zelandiae, Bipolaris ovariicola, Bipolaris panici-miliacei, Bipolaris papendorfii, Bipolaris sacchari, Bipolaris sakadehensis, Bipolaris sorghicola, Bipolaris subpapendorfii, Bipolaris tropicalis, Bipolaris urochloae, Bipolaris zeae, Puccinia 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, most preferably is Helminthosporium turcicum, Puccinia sorghi, Diplodia macrospora or Bipolaris maydis.
In one embodiment, the plant disease is a fungal disease. In a preferred embodiment, 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), Common Rust (caused by Puccinia sorghi), and Diplodia Leaf Streak (caused by Diplodia macrospora, also called Stenocarpella macrospora). Most preferably, the plant disease is Northern Corn Leaf Blight (NCLB).
In a preferred embodiment, the nucleic acid molecule of the invention is encoding a polypeptide capable of conferring to a plant (or increasing in a plant) resistance against Northern Corn Leave Blight (NCLB), i.e. resistance against Helminthosporium turcicum, in particular resistance against Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, or resistance against Helminthosporium turcicum races 0, 1 and/or N.
In one embodiment, the plant according to the invention 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, Beta vulgaris, Daucus pusillus, Daucus muricatus, Daucus carota, Eucalyptus grandis, Nicotiana sylvestris, Nicotiana tomentosiformis, Nicotiana tabacum, Nicotiana benthamiana, Solanum lycopersicum, Solanum 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 japonicas, Torenia fournieri, Allium cepa, Allium fistulosum, Allium sativum, Helianthus annuus, Helianthus tuberosus and Allium tuberosum, or any variety or subspecies belonging to one of the aforementioned plants, more preferably 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 aforementioned plants, or most preferably Hordeum vulgare, Sorghum bicolor, Zea mays, Triticum aestivum, Secale cereale, or any variety or subspecies belonging to one of the aforementioned plants.
In a preferred embodiment, the plant according to the invention is Zea mays.
Preferably, the nucleic acid molecule according to the present invention encodes a polypeptide which is a wall associated receptor-like kinases 1 (WAK RLK1) or functionally belongs to the family of wall associated receptor-like kinases 1.
In another aspect, provided is a vector comprising the nucleic acid molecule of the present invention. The vector may be a plasmid, a cosmid, a phage or an expression vector, a transformation vector, shuttle vector or cloning vector, it may be double or single stranded, linear or circular, or it may be a prokaryotic or eukaryotic host, either by integration into its genome or transforming extrachromosomally.
Also provided is an expression cassette comprising the nucleic acid molecule of the invention.
In one embodiment, in the vector or expression cassette, the nucleotide sequence of the invention is operably linked to regulatory element allowing expression of the nucleotide sequence in a plant cell. The plant cell may be infectable by a fungal pathogen or infected by a fungal pathogen. In a preferred embodiment, the plant cell is located in a leaf or a leaf tissue. The regulatory element may be a promoter (native, synthetic, core promoter or chimeric promoter), a terminator, an enhancer or a cis-acting element. Furthermore, the regulatory element may be heterologous to the nucleotide sequence operably linked to regulatory element.
Preferably, the nucleotide sequence of the invention is operably linked in an expression vector to one or more regulatory sequences which allow transcription and optionally expression in a prokaryotic or eukaryotic host cell. As an example, the nucleotide sequence may be under the control of a suitable promoter or a terminator. Suitable promoters may be promoters which are constitutively induced (see, for example, the 35S promoter from the “cauliflower mosaic virus” (Odell J T, Nagy F, Chua N-H (1985) “Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter.” Nature 313, 810-812 1985); other examples are the Actin promoter of Oryza sativa (SEQ ID NO: 43) or the EF1 promoter of Brachypodium distachyon (SEQ ID NO: 44). Particularly suitable promoters are those promoters which are pathogen-inducible (see, for example, the PR1 promoter from parsley (Rushton P J, Torres J T, Parniske M, Wernert P, Hahlbrock K und Somssich I E (1996) Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes. EMBO J. 15(20): 5690-5700). Particularly suitable pathogen-inducible promoters are synthetic or chimeric promoters which do not occur in nature, are composed of several elements and contain a minimum promoter as well as, upstream of the minimum promoter, at least one cis-regulatory element which act as the binding site for special transcription factors. Chimeric promoters are custom-designed and are induced by various factors or re-primed. Examples of such promoters can be found in WO2000/29592 and WO2007/147395. An example of a suitable terminator is the nos-terminator (Depicker A, Stachel S, Dhaese P, Zambryski P, Goodman H M (1982) Nopaline synthase: transcript mapping and DNA sequence. J Mol Appl Genet. 1(6): 561-73).
Further provided is a host cell comprising the nucleic acid molecule of the invention, or the vector of the invention, or the expression cassette of the invention.
The vector or the expression cassette may, for example, be introduced into the host cell by conjugation, mobilization, biolistic transformation, agrobacterium-conferred transformation, transfection, transduction, vacuum infiltration or electroporation. Methods of this type as well as methods for the preparation of the vectors described are familiar to the person skilled in the art (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory, 3rd Ed., 2001).
In one embodiment, the host cell may be a prokaryotic cell (for example, a bacterial cell). In another embodiment, the host cell may be a eukaryotic cell (for example, a plant cell or a yeast cell). Particularly preferred bacterial host cells are Agrobacterium tumefaciens, A. rhizogenes, and E. coli.
In another aspect, provided is a protein encoded by the nucleic acid molecule of the invention.
In yet another aspect, provided is a plant comprising the nucleic acid molecule of the invention, the vector of the invention, the expression cassette of the invention, or the protein of the invention. The plant may be a transgenic plant or a genetically edited plant. A part of the plant of the invention, plant cell of the plant of the invention and seed of the plant of the invention is also provided, wherein the seed comprising the nucleic acid molecule of the invention transgenically or endogenously, the vector of the invention, the expression cassette of the invention, or the polypeptide of the invention.
From WO2015/032494 which investigated and used introgression lines with Htn1 from Pepitilla, it is known that this restance locus is closely linked to genomic regions carrying linkage drag resulting in negative effects on one or more agronomic features. First investigation of the flanking region of HT2 indicated that this or similar linkage drag is not only present in Pepitilla donor for HtN1 introgression, but also in other donors for this Helminthosporium resistance locus like A619. Inter alia the linkage drag as part of the HT2 introgression can effect a difference in the flowering time, which is an important agronomic characteristic. It can directly and substantially influence the yield potential of a Zea mays plant. A delayed flowering time usually results in a reduced yield. Further linkage drag affecting the yield potential, in particular the silage yield potential, may be found distal and/or proximal of the Helminthosporium resistance locus on bin 8.06 in Zea mays. Flanking regions, closely linked to this resistance locus, might be carrier of the known linkage drag, however, these regions can be limited to an interval located between alleles of marker SYN14136 and marker MA0021 and/or an interval located between alleles of marker MA0022 and marker SYN4196 (
As an example, removal of linkage drag may be carried out by genetic recombination during a crossing process between two maize plants, wherein one parent maize plant carries the HT2-resistance locus. In addition to the use of conventional breeding techniques to produce a genetic recombination which has the result of replacing at least one of the donor intervals with linkage drag identified above with genomic sequences of the recurrent parent which are preferably free from unwanted genes, modern biotechnology offers the person skilled in the art many tools which can enable precise genetic engineering to be carried out. 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 (Gaj et al., 2013). These are artificial nuclease fusion proteins which are capable of cleaving double stranded nucleic acid molecules such as plant DNA and thus of producing double strand breaks at desired positions in the genome. By exploiting the cells own mechanisms for repairing induced double strand breaks, a homologous recombination or a “non-homologous end joining” can be carried out, which could lead to the removal of the intervals of the donor carrying linkage drag. Suitable target sequences in the genome for the recognition domain nucleases may be taken, for example, from the sequence information of the SNP markers (Table 2). However, a person skilled in the art is also able to identify other sequences, preferably within the defined flanking regions described above, which are suitable as target sequences for the recognition domains of the nucleases.
In another aspect, provided is a genetically edited or transgenic plant comprising the nucleic acid molecule of the invention, the vector of the invention or the expression cassette of the invention. The nucleic acid molecule may be a transgene or a modified/edited endogenous gene. A promoter may be operably linked to the nucleic acid molecule or nucleotide sequence for expression.
Also provided is a part or seed of the plant of the invention comprising the nucleic acid molecule of the invention, the vector of the invention or the expression cassette of the invention, wherein the seed comprises the nucleic acid molecule of the invention, the vector of the invention, the expression cassette of the invention. The nucleic acid molecule may be a transgene or a modified/edited endogenous gene.
According to another aspect, provided a method of identifying or selecting a plant, preferably a plant of the species Zea mays, having increased resistance to a plant disease caused by fungal pathogen, preferably by Helminthosporium turcicum, more preferably by Helminthosporium turcicum races 0, 1 and/or N, or a part, a cell or a seed thereof, comprising the following steps: (a) detecting in the plant, or part, cell or seed thereof (or in a sample of the plant, or part, cell or seed thereof), the presence of the 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%, 85%, 90%, 92%, 94%, 96%, 98%, 99% or 99.5% identity to the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, preferably over the full length, (ii) a nucleotide sequence encoding an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, 99% or 99.5% identity to the amino acid sequence set forth in SEQ ID NO: 3, preferably over the full length, (iii) a 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), preferably over the full length, and/or having at least 60%, 65%, 70%, 75%, 80%, 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 2; SEQ ID NO: 7) or to nucleotide positions 921-957 of the nucleotide sequence set forth in SEQ ID NO: 2 (Exon 2; SEQ ID NO: 7), preferably over the full length, and/or having at least 98%, 98.5%, 99% or 99.5% identity to nucleotide positions 23586-24632 of the nucleotide sequence set forth in SEQ ID NO: 1 (Exon 3; SEQ ID NO: 8) or to nucleotide positions 958-2004 of the nucleotide sequence set forth in SEQ ID NO: 2 (Exon 3; SEQ ID NO: 8), preferably over the full length, (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 to the positions 1-306 of amino acid sequence set forth in SEQ ID NO: 3, preferably over the full 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 amino acid sequence set forth in SEQ ID NO: 3, preferably over the full length, and/or having at least 98%, 98.5%, 99% or 99.5% identity to positions 320-668 of amino acid sequence set forth in SEQ ID NO: 3, preferably over the full length, wherein the nucleic acid molecule of any one of (i) to (iv) is encoding a polypeptide capable of conferring or increasing resistance to a plant disease caused by fungal pathogen, preferably by Helminthosporium turcicum, more preferably by Helminthosporium turcicum races 0, 1 and/or N, in a plant, preferably a plant of the species Zea mays, in which the polypeptide is expressed, and wherein preferably the nucleic acid molecule of any one of (i) to (iv) encodes a polypeptide which is or belongs functionally to the family of wall associated receptor-like kinases 1 (WAK RLK1); or detecting in the plant, or part, cell or seed thereof (or in a sample of the plant, or part, cell or seed thereof), the presence of polypeptide encoded by the nucleic acid molecule of the present invention as described above or a nucleotide sequence of any one of (i) to (iv), and (b) identifying or selecting the plant in which or in whose part, cell or seed the nucleic acid molecule as defined in (a) is present, as having a resistance or an increased resistance to a plant disease caused by a fungal pathogen, preferably by Helminthosporium turcicum, more preferably by Helminthosporium turcicum races 0, 1 and/or N.
The method may comprise the additional step of obtaining a nucleic acid sample from the plant or part, cell or seed and detecting the sequence in the sample. For example, the step of obtaining the nucleic acid sample from the plant, or part or seed thereof, may be carried out prior to the detection step (a).
The detection of a nucleotide sequence may be carried out by a hybridization method, using an oligonucleotide probe. The conditions of hybridization can appropriately be selected 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.
Alternatively, the detection of a gene may be carried out by means of a DNA amplification method, such as PCR, using respective primers. When the detection is carried out by PCR method, PCR conditions can appropriately be selected depending on the factors such as the Tm value of the primer used and the length of the amplified region to be detected. The detection can be carried out by amplifying the target by PCR and confirming the presence or absence of a 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 a reaction mixture of nucleic acid amplification to agarose gel electrophoresis; thereafter, staining the gel with an appropriate nucleic acid staining reagent such as ethidium bromide, SYBER Green I or the like; and detecting the presence or absence of the bands resulting from irradiation with ultraviolet rays. The bands may be detected by visual observation, or they may be detected by using, for example, a fluorescent image analyzer, or the like.
Further provided is, a method or process of identifying a plant, preferably a plant of the species Zea mays, having increased resistance to a plant disease caused by fungal pathogen, Helminthosporium turcicum, more preferably by Helminthosporium turcicum races 0, 1 and/or N, or a part, having increased resistance to a plant disease caused by Helminthosporium turcicum, preferably by Helminthosporium turcicum races 0, 1 and/or N and comprises the nucleic acid molecule of the invention endogenously, a cell or a seed thereof, the method or process comprising detecting in the plant alleles of at least two markers, wherein at least one of said markers is on or within the chromosomal interval between SYN14136 and the nucleic acid molecule of the invention, and at least one of said markers is on or within the chromosomal interval between the nucleic acid molecule of the invention and SYN4196. In a preferred embodiment, the method or process comprising detecting in the plant alleles of at least two markers, wherein at least one of said markers is on or within the chromosomal interval between PZE108093423 and the nucleic acid molecule of the invention, and at least one of said markers is on or within the chromosomal interval between the nucleic acid molecule of the invention and SYN4196.
In another aspect, provided is a method of identifying an allele of a resistance gene conferring or increasing resistance to a plant disease caused by a fungal pathogen, preferably by Helminthosporium turcicum, more preferably by Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, in Zea mays and preferably encoding a polypeptide which is or belongs functionally to the family of wall associated receptor-like kinases 1 (WAK RLK1), wherein the method comprises the following steps: (a) conducting sequence comparison using (i) at least one coding nucleotide sequence isolated from a Zea mays genotype, wherein the nucleotide sequence preferably maps to bin 8.05 resistance locus or to bin 8.06 resistance locus, and (ii) as reference sequence, a nucleotide sequence of the invention, preferably the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 or the nucleotide sequence encoding the amino acid sequence set forth 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 invention, preferably the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 or the nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 3, or a part 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 fungal pathogen, preferably by Helminthosporium turcicum, more preferably by Helminthosporium turcicum races 0, 1 and/or N, in Zea mays in which the polypeptide is expressed, and preferably maps to bin 8.05 resistance locus or to bin 8.06 resistance locus and preferably encoding a polypeptide which is or belongs functionally to the family of wall associated receptor-like kinases 1 (WAK RLK1); and (b) identifying the allele, if the sequence comparison reveals (i) a sequence identity on nucleotide level 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, preferably over the full length, and/or of at least 60%, 65%, 70%, 75%, 80%, 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 or to nucleotide positions 921-957 of the nucleotide sequence set forth in SEQ ID NO: 2, preferably over the full length, and/or of at least 98%, 98.5%, 99% or 99.5% identity to nucleotide positions 23586-24632 of the nucleotide sequence set forth in SEQ ID NO: 1 or to nucleotide positions 958-2004 of the nucleotide sequence set forth in SEQ ID NO: 2, preferably over the full length, and/or (ii) a sequence identity of encoded amino acid level of at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, 99% or 99.5% identity to the positions 1-306 of amino acid sequence set forth in SEQ ID NO: 3, preferably over the full length, and/or of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, 99% or 99.5% identity to positions 307-319 of amino acid sequence set forth in SEQ ID NO: 3, preferably over the full length, and/or of at least 98%, 98.5%, 99% or 99.5% identity to positions 320-668 of amino acid sequence set forth in SEQ ID NO: 3, preferably over the full length.
Preferably, the consensus sequence is derived from a set of at least two nucleotide sequences wherein one nucleotide sequence is the nucleotide sequence of the invention, preferably the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 or the nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 3, or a part thereof, and wherein a further nucleotide sequence is selected from the group consisting of the nucleotide sequence set forth in SEQ ID NO: 9 or a part thereof, the nucleotide sequence set forth in SEQ ID NO: 11 or a part thereof, the nucleotide sequence set forth in SEQ ID NO: 13 or a part thereof, the nucleotide sequence set forth in SEQ ID NO: 23 or a part thereof, a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 10 or a part thereof, a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 12 or a part thereof, a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 14 or a part thereof, or a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 24 or a part thereof.
The at least one coding nucleotide sequence isolated from a Zea mays genotype of step (a) (i) can be derived from a plant sequence database or from a gene bank where seed samples of various Zea mays genotype are deposited. Plant sequence databases or gene banks are known in the art.
The method of 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 fungal pathogen may further comprise, e.g. as step (c), the step of determining the resistance level of the plant against the fungal pathogen caused by the identified allele.
A nucleic acid molecule comprising or consisting of a nucleotide sequence encoding an allele of a resistance gene (or potential new allele) identified by the method of identifying described above is also provided. Said allele may be used in a method of producing a plant, preferably a plant of the species Zea mays, or in a method for conferring/increasing resistance to a plant disease which is caused by a fungal pathogen such as Helminthosporium turcicum, preferably by Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N. In one embodiment, the method according to the invention of producing a plant or the method according to the invention for conferring/increasing resistance to a plant disease further comprises in step (a) the introduction of at least one nucleic acid molecule comprising or consisting of a nucleotide sequence encoding the new resistance gene into the at least one cell of the plant.
In another aspect, provided is a method for conferring or increasing resistance to a plant disease caused by a fungal pathogen, preferably by Helminthosporium turcicum, more preferably by Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, in a plant, preferably a plant of the species Zea mays, comprising the following steps: (a) introducing into at least one cell of the plant the nucleic acid molecule of the present invention, including for example a nucleic acid molecule comprising or consisting of a nucleotide sequence of the identified allele, the expression cassette of the invention or the vector of the invention, (b) regenerating the plant from the at least one cell, and (c) causing expression of the nucleic acid molecule in the plant.
In a preferred embodiment of the method for conferring or increasing resistance to a plant disease caused by a fungal pathogen, step (a) results in the modification of an endogenous nucleic acid molecule conferring susceptibility to a plant disease caused by a fungal pathogen, preferably by Helminthosporium turcicum, more preferably by Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, preferably mapping to bin 8.05 resistance locus or to bin 8.06 resistance locus on chromosome 8 of Zea mays, and preferably encoding a polypeptide which is or belongs functionally to the family of wall associated receptor-like kinases 1 (WAK RLK1), wherein the modification converts the endogenous nucleic acid molecule into the nucleic acid molecule of the present invention conferring resistance to a plant disease caused by a fungal pathogen, preferably by Helminthosporium turcicum, more preferably by Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, if expressed.
In another preferred embodiment of the method for conferring or increasing resistance to a plant disease caused by a fungal pathogen, step (a) results in the modification of an endogenous nucleic acid molecule conferring resistance to a plant disease caused by a fungal pathogen, preferably by Helminthosporium turcicum, more preferably by Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, preferably mapping to bin 8.05 resistance locus or to bin 8.06 resistance locus on chromosome 8 of Zea mays, and preferably encoding a polypeptide which is or belongs functionally to the family of wall associated receptor-like kinases 1 (WAK RLK1), wherein the modification converts the endogenous nucleic acid molecule into the nucleic acid molecule of the present invention conferring increased resistance to a plant disease caused by a fungal pathogen, preferably by Helminthosporium turcicum, more preferably by Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, if expressed.
Further provided is a method for increasing resistance to a plant disease caused by a fungal pathogen, preferably by Helminthosporium turcicum, more preferably by Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, in a plant of the invention, comprising the steps of reducing the level of expression of the nucleic acid molecule of the present invention, including for example a nucleic acid molecule comprising or consisting of a nucleotide sequence of the identified allele, the expression cassette of the invention or the vector of the invention, in the plant or at least one cell of the plant, preferably compared to the expression level of the endogenous gene in a resistant wild type plant. Such resistant wild type plant is for instances selected from the lines A619HT2, B37HT2 or B73HT2. Reducing can be conducted transiently or durably, preferably as preventive measure if infestation by the fungal pathogen in expected e.g. due to particular environmental conditions which drives the distribution of the fungal pathogen. A person skilled in the art is knowing very well various methodologies to reduce the expression of gene in plant. Durable reduction of expression level can be achieved e.g. by modulating the promoter or other regulatory elements by means of random or targeted mutagenesis or by stable integration of an expression cassette allowing the expression of double-stranded RNA, single-stranded antisense RNA or a hairpin DNA, which are able to silence as interfering RNA the mRNA encoded by the nucleic acid molecule of the invention. Such inhibitory RNA molecule, preferably in form of siRNA molecules, can also be used for transient reduction of the level of expression if applied to the plants as spray and taken up actively or passively by the plant cells, preferably cells of leaves.
In another aspect, provided is a method for producing a plant (or part, cell or seed thereof), preferably a plant of the species Zea mays, having (increased) resistance to a plant disease caused by fungal pathogen, preferably by Helminthosporium turcicum, more preferably by Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, or a part, a cell or a seed thereof, comprising the following steps: (a) introducing into the plant or at least one cell of the plant the nucleic acid molecule of the present invention, including for example a nucleic acid molecule comprising or consisting of a nucleotide sequence of the identified allele, the expression cassette of the invention or the vector of the invention, (b) optionally, regenerating the plant from the at least one cell, and (c) causing expression of the nucleic acid molecule in the plant or part thereof.
In a preferred embodiment of the method 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 susceptibility to a plant disease caused by a fungal pathogen, preferably by Helminthosporium turcicum, more preferably by Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, preferably mapping to bin 8.05 resistance locus or to bin 8.06 resistance locus on chromosome 8 of Zea mays, and preferably encoding a polypeptide which is or belongs functionally to the family of wall associated receptor-like kinases 1 (WAK RLK1), wherein the modification converts the endogenous nucleic acid molecule into the nucleic acid molecule of the present invention conferring resistance to a plant disease caused by a fungal pathogen, preferably by Helminthosporium turcicum, more preferably by Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, if expressed.
In another preferred embodiment of the method 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 resistance to a plant disease caused by a fungal pathogen, preferably by Helminthosporium turcicum, more preferably by Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, preferably mapping to bin 8.05 resistance locus or to bin 8.06 resistance locus on chromosome 8 of Zea mays, and preferably encoding a polypeptide which is or belongs functionally to the family of wall associated receptor-like kinases 1 (WAK RLK1), wherein the modification converts the endogenous nucleic acid molecule into the nucleic acid molecule of the present invention conferring increased resistance to a plant disease caused by a fungal pathogen, preferably by Helminthosporium turcicum, more preferably by Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, if expressed.
Preferably, the method is for producing a plant (or part, cell or seed thereof) having increased resistance to a plant disease caused by fungal pathogen, such as Helminthosporium turcicum, preferably by Helminthosporium turcicum races 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 that has increased resistance compared to a reference plant.
Preferably, the reference plant is a plant of the same species, more preferably the reference plant is a wild-type plant of the same species or a plant isogenic to the plant selected in step (d), expect for the nucleic acid molecule of the present invention that has introduced in step (a) or modified during step (a) as described above.
Preferably, in the methods of the invention, the nucleic acid molecule of the invention is expressed in the plant or part thereof in an amount and/or period sufficient to increase or confer resistance to a plant disease caused by a fungal pathogen in the plant.
In another aspect, provided is a plant produced by the methods of the invention, or progeny, fruit, or seed thereof. Preferably, the plant or progeny, fruit, or seed thereof comprises the nucleic acid molecule of the invention, or the vector of the invention, or the protein of the invention, and/or the cell of the invention.
In another aspect, provided is the use of at least one nucleic acid molecule of the invention, including for example a nucleic acid molecule comprising or consisting of a nucleotide sequence of the identified new allele of a resistance gene, in the production of a plant having increased resistance to a plant disease caused by a fungal pathogen, preferably Helminthosporium turcicum, more preferably Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N. The plant produced may be a genetically edited or a transgenic plant.
Further provided is a method for controlling infestation of a fungal pathogen, preferably Helminthosporium turcicum, more preferably 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 on agricultural and horticultural fields, and (b) causing expression of the nucleic acid molecule of the present invention, including for example a nucleic acid molecule comprising or consisting of a nucleotide sequence of the identified allele, the expression cassette of the invention or the vector of the invention, in the plants.
Another aspect of the invention is an oligonucleotide having a length of at least 15, 16, 17, 18, 19 or 20, preferably at least 21, 22, 23, 24 or 25, more preferred at least 30, 35, 40, 45, 50, 100, 200, 300 or 500 nucleotides, wherein the oligonucleotide is able to hybridize or anneal to (i) the nucleic acid molecule of the invention, (ii) the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 5, or (iii) nucleic acid molecule complementary to (i) or (ii). Preferably, the oligonucleotide comprises a nucleotide sequence having an identity of at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5% or 100% to the nucleotide sequence of the nucleic acid molecule of (i), (ii) or (iii) to which the oligonucleotide is able to hybridize or anneal.
Further provided is a pair of oligonucleotides or a kit comprising said oligonucleotides, wherein the oligonucleotides are suitable to anneal as forward primer and reverse primer to a region in the plant genome, preferably the Zea mays genome, which shows a cosegregation, preferably a perfect cosegregation, with the nucleic acid molecule of the invention. Preferably, the pair comprises one or two oligonucleotides of the invention.
Preferably, the kit comprises one, two or more oligonucleotides of the invention In a further aspect, provided is the use of at least one oligonucleotide as a molecular marker or part thereof in the method of identifying or selecting a plant, preferably a plant of the species Zea mays, having increased resistance to a plant disease caused by fungal pathogen, preferably by Helminthosporium turcicum, more preferably by Helminthosporium turcicum races 0, 1 and/or N, or a part, a cell or a seed thereof, or in the method of identifying an allele of a resistance gene conferring or increasing resistance to a plant disease caused by a fungal pathogen, preferably by Helminthosporium turcicum, more preferably by Helminthosporium turcicum races 0, 1 and/or N, in Zea mays, wherein the molecular marker is able to detect at least one single nucleotide polymorphism, deletion or insertion diagnostic for the nucleic acid molecule of the present invention and/or comprises the oligonucleotide as described above and/or is the pair of oligonucleotides or the kit as described above. Such single nucleotide polymorphism, deletion or insertion can be directly derived from the sequence alignment shown in
In another aspect, provided is a method for detecting the presence or absence of the nucleic acid molecule of the invention, including for example a nucleic acid molecule comprising or consisting of a nucleotide sequence of the identified allele, in a plant, preferably a plant of the species Zea mays, comprising the following steps: (a) isolating DNA from at least one cell of the plant, and (b) using a molecular marker to detect the presence or absence of the nucleic acid molecule of the invention, wherein the molecular marker is able to detect at least one single nucleotide polymorphism, deletion or insertion diagnostic for the nucleic acid molecule of the present invention and/or comprises the oligonucleotide as described above and/or is the pair of oligonucleotides or the kit as described above.
In one aspect, provided is the use of at least one or at least two oligonucleotide(s) as a molecular marker or part thereof in method or process of identifying a plant as described above in a method or process for the elimination of linkage drag close linked to the nucleic acid molecule of the invention for detecting the presence or absence of an A619HT2 or A619HT3 derived interval located between alleles of marker SYN14136 and marker MA0021 or an A619HT2 or A619HT3 derived interval located between alleles of marker MA0022 and marker SYN4196. In a preferred embodiment, at least one oligonucleotide is used as molecular marker or part thereof in a method or process for the elimination of linkage drag close linked to the nucleic acid molecule of the invention for detecting the presence or absence of an A619HT2 or A619HT3 derived interval located between alleles of marker SYN14136 and marker PZE108077560, an A619HT2 or A619HT3 derived interval located between alleles of marker PZE108093423 and marker MA0021, and/or an A619HT2 or A619HT3 derived interval located between alleles of marker MA0022 and marker SYN4196.
In another aspect, provided is a method for detecting the presence of the nucleic acid molecule of the invention, including for example a nucleic acid molecule comprising or consisting of a nucleotide sequence of the identified allele, in a plant, preferably a plant of the species Zea mays, comprising the following steps: (a) isolating DNA from at least one cell of the plant, and (b) using a molecular marker to detect the presence or absence of the nucleic acid molecule of the invention, wherein the molecular marker is able to detect at least one single nucleotide polymorphism, deletion or insertion diagnostic for the nucleic acid molecule of the present invention and/or comprises the oligonucleotide as described above and/or is the pair of oligonucleotides or the kit as described above. Such single nucleotide polymorphism, deletion or insertion can be directly derived from the sequence alignment shown in
The term “resistance” or “resistant” as regards a pathogen should be understood to mean the ability of a plant, plant tissue or plant cell to resist the damaging effects of the pathogen and extends from a delay in the development of disease to complete suppression of the development of the disease. The resistance may be complete or partial and may be specific or non-specific to the pathogen race. A conferred resistance may be a newly inherited resistance or an increase in a partial resistance which is already extant.
Resistance may be quantified by methods known in the art. For example, resistance to Helminthosporium turcicum may be quantified by determining classification scores using phenotyping experiments in accordance with the scheme shown in the Table 4 below. For example, a Helminthosporium turcicum-resistant maize plant in the meaning of the invention exhibits an “increased resistance” to H. turcicum by at least 1 classification score, preferably by at least 2 classification scores or at least 3 classification scores, and most preferably by at least 4 classification scores. Preferably, a maize plant in accordance with the invention exhibits resistance to at least one race of Helminthosporium turcicum which does not correspond to the known race specificity known in the prior art. In a particularly preferred embodiment, a maize plant in accordance with the invention is resistant to all known races of Helminthosporium turcicum, i.e. the conferred resistance is not race-specific and may be particularly advantageous in the formation of a broad resistance to Helminthosporium turcicum.
H. turcicum inoculation (from the Deutsche Maiskomitee
The term “hybridize” or “hybridization” should be understood to mean a procedure in which a single stranded nucleic acid molecule agglomerates with a nucleic acid strand which is as complementary as possible, i.e. base-pairs with it. Examples of standard methods for hybridization have been described in 2001 by Sambrook et al. Preferably, this should be understood to mean that at least 60%, more preferably at least 65%, 70%, 75%, 80% or 85%, particularly preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the bases of the nucleic acid molecule undergo base pairing with the nucleic acid strand which is as complementary as possible. The possibility of such agglomeration depends on the stringency of the hybridization conditions. The term “stringency” refers to the hybridization conditions. High stringency is when base pairing is more difficult, low stringency is when base pairing is easier. The stringency of the hybridization conditions depends, for example, on the salt concentration or ionic strength and the temperature. In general, the stringency can be increased by raising the temperature and/or by reducing the salt content. The term “stringent hybridization conditions” should be understood to mean those conditions under which a hybridization takes place primarily only between homologous nucleic acid molecules. The term “hybridization conditions” in this respect refers not only to the actual conditions prevailing during actual agglomeration of the nucleic acids, but also to the conditions prevailing during the subsequent washing steps. Examples of high stringent hybridization conditions are conditions under which primarily only those nucleic acid molecules that have at least 90% or at least 95% sequence identity undergo hybridization. Such high stringent hybridization conditions are, for example: 4×SSC at 65° C. and subsequent multiple washes in 0.1×SSC at 65° C. for approximately 1 hour. The term “high stringent hybridization conditions” as used herein may 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 and subsequently washing twice with 2×SSC and 0.1% SDS at 68° C. Preferably, hybridization takes place under stringent conditions. Less stringent hybridization conditions are, for example: hybridizing in 4×SSC at 37° C. and subsequent multiple washing in 1×SSC at room temperature.
The present invention encompasses nucleic acid molecules comprising or consisting of a nucleotide sequence encoding a protein, said protein being derived from the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or from the amino acid sequence set forth in SEQ ID NO: 3 by way of substitution, deletion and/or addition of one or more amino acid(s). Herein, the term “one or more amino acid(s)” refers to, for example, 1 to 50, 1 to 40, 1 to 30, or 1 to 20, preferably 1 to 10, more preferably 1 to 7, further preferably 1 to 5, and particularly preferably 1, 2, or 3 amino acids. “Operably linked” means linked in a common nucleic acid molecule in a manner such that the linked elements are positioned and orientated with respect to each other such that transcription of the nucleic acid molecule can take place. A DNA which is operably linked with a promoter is under the transcriptional control of this promoter. “Introducing” in the meaning of the present invention includes stable integration by means of transformation including Agrobacterium-mediated transformation, transfection, microinjection, biolistic bombardment, insertion using gene editing technology like CRISPR systems (e.g. CRISPR/Cas, in particular CRISPR/Cas9 or CRISPR/Cpf1), CRISPR/CasX, or CRISPR/CasY), TALENs, zinc finger nucleases or meganucleases, homologous recombination optionally by means of one of the above mentioned gene editing technology including preferably a repair template, modification of endogenous gene using random or targeted mutagenesis like TILLING or above mentioned gene editing technology, etc. The term “introducing” may or may not encompass the introgression using conventional breeding.
The term “transgenic”, “transgenical” or “transgene” is understood to mean that the respective gene is an exogenous gene that was introduced into the plant. The exogenous gene may be derived from a species other than the plant species into which it is introduced. Alternatively, the respective gene may be a gene already present in the plant species into which it is introduced, so that one or more additional copies of said gene are present as a result introducing the transgene. A “transgenic plant” is a plant into the genome of which at least one polynucleotide, preferably a heterologous polynucleotide, has been integrated. Preferably, the polynucleotide has been integrated in a stable manner, which means that the integrated polynucleotide remains stable in the plant, is expressed and can also be stably inherited to descendants. The term “heterologous” means that the introduced polynucleotide originates, for example, from a cell or an organism with another genetic background of the same species or from another species, or is homologous with the prokaryotic or eukaryotic host cell, but then is localized in a different genetic environment and thus is different from any possible corresponding naturally occurring polynucleotide. A heterologous polynucleotide can be present in addition to a corresponding endogenous gene.
Plant “organs” are leaves, plant stems, stems, roots, vegetative buds, meristems, embryos, anthers, ovulae or fruit. Plant “parts” can mean a fusion of several organs, for example a flower or a seed or a part of an organ, for example a cross segment from the stem. Examples of plant “tissues” are callus tissue, storage tissue, meristematic tissue, embryogenic tissue, leaf tissue, bud tissue, root tissue, plant tumour tissue or reproductive tissue. The term “cells” should be understood to mean isolated plant cells with a cell wall or aggregates thereof or protoplasts, for example.
The term “genetically edited” means that an endogenous gene is modified e.g. by means of random mutagenesis, TILLING or gene editing technology. For example, according to the invention, the endogenous gene of a plant may be modified to confer resistance (or increased resistance) to a plant disease caused by a fungal pathogen. Genetic modification may, for example, be achieved using methods of random or targeted mutagenesis (such as gene editing), or homologous recombination (optionally, supported by gene editing tools) or combinations thereof.
As used herein, “a modification”, means that the genetic sequence has changed by at least one nucleotide. This can occur by replacement of at least one nucleotide and/or a deletion of at least one nucleotide and/or an insertion of at least one nucleotide, as long as it results in a total change of at least one nucleotide compared to the nucleotide sequence before modification, thereby allowing the identification of the modification, e.g. by techniques such as sequencing or PCR analysis and the like, of which the skilled person will be well aware.
The term “allele” refers to one or two or more nucleotide sequences at a specific locus in the genome. A first allele is on one chromosome, a second allele on the sister chromosome at the same position. If the two alleles are different, they are heterozygous, and if they are the same, they are homozygous. Various alleles of a gene (gene alleles) differ in at least one SNP. Various alleles of a resistance gene may either confer resistance, possibly different level of resistance to a plant against a fungal pathogen, i.e. causes differ types of phenotypes of the plant in response to infestation with fungal pathogen, or constitute a variant of the gene which is not able to confer resistance, i.e. the resulting plant phenotype is susceptible to a fungal pathogen.
According to the present invention, the term “regulatory sequence” means a nucleotide sequence which influences the specificity and/or strength of expression, for example insofar as the regulatory sequence confers a specific tissue specificity. A regulatory sequence of this type may be localized upstream of the transcription initiation point of a minimum promoter, but also downstream thereof, for example in a transcribed but not translated leader sequence or within an intron.
A “molecular marker” or “marker” is a nucleotide sequence which is used as a reference or orientation point. A marker for recognizing a recombination event should be suitable for monitoring differences or polymorphisms in a plant population. For markers, these differences are on a DNA level and, for example, are polynucleotide sequence differences such as, for example, SSRs (simple sequence repeats), RFLPs (restriction fragment length polymorphisms), FLPs (fragment length polymorphisms) or SNPs (single nucleotide polymorphisms). The markers may be derived from genomic or expressed nucleic acids such as spliced RNA, cDNA or ESTs and may be based on nucleic acids which are used as probes or primer pairs and as such are suitable for amplifying a sequence fragment using PCR-based methods. Markers which concern genetic polymorphisms between parts of a population can be detected using established methods from the prior art (An Introduction to Genetic Analysis. 7th Edition, Griffiths, Miller, Suzuki et al., 2000). These include, for example: DNA sequencing, PCR-based, sequence-specific amplification, assaying of RFLPs, assaying of KASP, assaying of polynucleotide polymorphisms using allele-specific hybridization (ASH), detection of SSRs, SNPs or AFLPs. Methods for detecting ESTs (expressed sequence tags) and RAPD (randomly amplified polymorphic DNA) are also known. Depending on the context, the term “marker” in the description may also mean a specific chromosome position in the genome of a species where a specific marker (for example SNP) can be found.
The terms “distal” and “proximal” describe the position of a chromosomal interval or a genetic segment in relation to a specific reference point (for example a specific polynucleotide, another chromosomal interval or a gene) on a whole chromosome; “distal” means that the interval or the segment is localized on the side of the reference point distant from the chromosome centromere, and “proximal” means that the interval or the segment is localized on the side of the reference point close to the chromosome centromere. “closely linked” means two loci, two intervals, two genetic segments (e.g. resistance gene and flanking regions) or two markers (marker loci) which are less than 15 cM, less than 12 cM, less than 10 cM, less than 8 cM, 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 distant from each other, established using the IBM2 neighbors 4 genetic map which is publicly available on the Maize GDB website, or which are less than 50 Mbp (mega base pairs), less than 40 Mbp, less than 30 Mbp, less than 25 Mbp, less than 20 Mbp, less than 15 Mbp, or less than 10 Mbp distant from each other.
The term “interval” or “chromosomal interval” means a continuous linear segment on a genomic DNA which is present in an individual chromosome in a plant or on a chromosome fragment and which is usually defined through two markers which represent the end points of the interval on the distal and proximal side. In this regard, the markers which define the ends of the interval may themselves also be a part of the interval. Furthermore, two different intervals might overlap. In the description, an interval is specified by the statement “between marker A and marker B”. An end marker of an interval may also be localized in a defined marker region to one side of the interval. A marker region is then defined by providing two flanking markers and constitutes a chromosomal segment on which more markers might be located, in addition to the flanking markers. Flanking markers determine the end points of a marker region and are themselves still a part of the marker region. If both end markers of an interval are markers in different marker regions on both sides of an interval, the description specifies an interval by stating “between a marker in a marker region X which is flanked by the markers C and D and a marker in a marker region Y which is flanked by markers E and F”. A marker region may extend over up to 500 000 base pairs (bp), and can preferably be between 100 000 and 400 000 bp in size, or can particularly preferably be between 140 000 and 315 000 bp in size.
The term “introgression” as used in connection with the present invention means the transfer of at least one desired gene allele on a genetic locus of a genetic background into another. As an example, an introgression of a desired gene allele at a specific locus may be transferred to a descendant by sexual crossing between two parents of the same species. Alternatively, for example, the 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 the descendants, which then comprise the desired gene allele, can then be backcrossed again with a line which comprises a preferred genetic background and can be selected for the desired gene allele. The result is fixing of the desired gene allele in a selected genetic background.
A “Locus” is a position on a chromosome where one or more genes are found which cause an agronomic feature or influence one. In particular, “locus” as used here means the HT2-resistance locus which confers resistance against the pathogen Helminthosporium turcicum or at least against a race of Helminthosporium turcicum.
The term “allele” refers to one or two or more nucleotide sequences at a specific locus in the genome. A first allele is on a chromosome, a second on a second chromosome at the same position. If the two alleles are different, they are heterozygous, and if they are the same, they are homozygous. Various alleles of a gene (gene alleles) differ in at least one SNP.
Depending on the context of the description, an allele also means a single SNP which, for example, allows for a distinction between the resistance donor and recurrent parent.
The following examples, including the experiments conducted and the results achieved, are provided for illustrative purposes only and are not construed as limiting the present invention.
a. QTL Mapping and Development of Recombinants
The donor line A619HT2 was crossed and backcrossed with the line RP1 to create a near isogenic line (NIL, RP1HT2A) with the main fragment of the original donor A619HT2 on chromosome 8 and very few other small donor regions. This NIL RP1HT2A was crossed with its recurrent parent RP1 to build up a F2 population. The same was done for RP2×RP2HT3A (original donor was here A619HT3). The recurrent parents RP1 and RP2 were susceptible to NCLB (Scores 7-9; cf. Table 4), while the donor lines A619HT2 and A619HT3 are resistant (scores 1-3). The scores of NILs RP1HT2A and RP2HT3A are 2-3 and 1-2, respectively.
The F2 populations were planted in the field with 720 individuals at the location Pocking, Germany. QTL mapping resulted in a peak (LOD value for population with RP2HT3A=168.77; LOD value for population with RP1HT2A=44.02) for both populations on chromosome 8 (1.1 cM and 3.5 Mbp frame). No other significant peak was detected. Recombinant plants (No. total˜2000) were developed in several generations until F11 status. QTL mapping in F2 and further fine mapping with recombinants narrowed down the chromosomal region down to a physical interval of 490 kbp (genetic interval=0.2 cM). This interval includes the RLK1 gene.
b. Molecular Analysis of Target Region
From the lines RP1HT2A and RP2HT3A non-gridded BAC libraries were developed and screened with 7 probes from the target locus. For the RLK1 candidate gene region a contiguous BAC contig could be developed for both donor lines. Sequence analysis revealed that the donor line A619HT2 and A619HT3 are identical for the target region (in 1 Mbp). The candidate gene RLK1 cDNA sequence shows 97 polymorphisms (DNA level; incl. SNPs and Indels/Deletions) and 61 amino acid changes (protein level) between the lines harboring the HTN1 of WO 2015/032494 A2 and the HT2 allele. Out of the 97 polymorphisms, only 14 single nucleotide polymorphisms (SNPs) result in silent amino acid exchanges which probably do not influence the activity of the resistance gene. All other polymorphisms change the structure of the protein significantly by substitution, addition or deletion. Table 3 shows 15 amino acid exchanges for which a strong effect on the protein structure is predicted. Among these, multiple genotype specific additional amino acids have been identified. Compared to RLK1 cDNA sequences set forth in SEQ ID NO: 11 and 13 from the donor line disclosed in WO 2011/163590 A1, A619HT2 and A619HT3 differ in 31 nucleotides and 12 amino acids.
c. Functional Validation of the HT2 Allele of RLK1
(i) Functional Validation Using EMS Mutagenesis:
An EMS-mutagenized population from RP2HT3A was developed. The exonic regions 1 and 3 from RLK1 were screened and 3 positive mutants harbouring an amino acid change were detected (SEQ ID NOs: 16, 18, 20, 46, 48, 50, 52, 54, 56 and 58). In RLK cDNA of Mutant WVE16-92125-001 G at position 1625 replaced by A (see also SEQ ID NO: 15), leading to an amino acid exchange from Gly to Asp at position 542 (see also SEQ ID NO: 16); in RLK cDNA of Mutant WVE16-92149-012 C at position 95 replaced by T (see also SEQ ID NO: 17), leading to an amino acid exchange from Pro to Leu at position 32 (see also SEQ ID NO: 18); in RLK cDNA of Mutant WVE16-92168-005 G at position 115 replaced by A (see also SEQ ID NO: 19), leading to an amino acid exchange from Ala to Thr at position 39 (see also SEQ ID NO: 20); in RLK cDNA of Mutant WVE16-92168-024_WVE17-68687-013 G at position 73 replaced by A (see also SEQ ID NO: 45), leading to an amino acid exchange from Ala to Thr at position 25 (see also SEQ ID NO: 46); in RLK cDNA of Mutant WVE17-68655-008 C at position 301 replaced by T (see also SEQ ID NO: 47), leading to an amino acid exchange from Pro to Ser at position 101 (see also SEQ ID NO: 48); in RLK cDNA of Mutant WVE17-68625-014 G at position 715 replaced by A (see also SEQ ID NO: 49), leading to an amino acid exchange from Val to Ile at position 239 (see also SEQ ID NO: 50); in RLK cDNA of Mutant WVE17-68611-006 T at position 862 replaced by A (see also SEQ ID NO: 51), leading to an amino acid exchange from Phe to Ile at position 288 (see also SEQ ID NO: 52); in RLK cDNA of Mutant WVE17-68696-002 A at position 929 replaced by G (see also SEQ ID NO: 53), leading to an amino acid exchange from Lys to Arg at position 310 (see also SEQ ID NO: 54); in RLK cDNA of Mutant WVE17-68656-011 C at position 1289 replaced by T (see also SEQ ID NO: 55), leading to an amino acid exchange from Thr to Ile at position 430 (see also SEQ ID NO: 56); in RLK cDNA of Mutant WVE17-68630-001 G at position 1826 replaced by A (see also SEQ ID NO: 57), leading to an amino acid exchange from Cys to Tyr at position 609 (see also SEQ ID NO: 58). After selfing of these mutants, they have been evaluated in the field and greenhouse.
d. Expression Analysis:
The expression analysis of RLK1 in RP1, RP1HT2A, RP2 and RP2HT3A in non-infected and infected leaf material showed a similar expression in the NILs RP1HT2A and RP2HT3A. The expression is down-regulated in the infected leaf material. This response to infection could also be shown for the HTN1 allele of RLK1.
In order to identify a relevant region for the resistance reaction to the pathogen Helminthosporium turcicum, the following analysis are carried out: Bioinformatic analysis of re-sequencing of the RLK1 gene in different donor lines and recurrent parents reveals the amino acid region relevant for the resistance reaction. Therefore, genomic sequence primer pairs for RLK1 have been developed to cover the exonic regions. Exon 1 could only be covered partially, and Exon 2 and 3 completely. The developed amplicons were amplified and sequenced in 96 genotypes with the PACBio-sequencing technique. The sequences were assembled to consensus sequences per genotype. For some genotypes, multiple consensus-sequences were obtained. The consensus sequence with the highest amount of sequencing reads was chosen for an assembly of all 96 genotypes. The haplotypes were determined according to the exonic region/parts. For Exon 1, 12 different haplotypes; for Exon 2, 7 different haplotypes; and for Exon 3, 9 different haplotypes were detected. The genetic distance was calculated within the software Lasergene MegAlign (DNASTAR, Inc.). The different haplotypes were assembled on DNA and Protein sequence level (see Table 5). As a result, the Exon 1 and 2 regions seems to be highly variable for the gene and harbor the WAK-associated domains. This protein part is located in the intercellular space and could interact with fungal proteins. Variances in these two exons are interesting candidate base pairs for this interaction. On this basis, it is possible to identify haplotypes of Exon 1 and 2 in new alleles of RLK1 which are able to confer or increase resistance at least to the pathogen Helminthosporium turcicum.
Furthermore, phenotyping of different donor lines, near isogenic lines and recurrent parents and haplotype analysis of the RLK1 locus as well as expression analysis of RLK1 in different donor lines, near isogenic lines and recurrent parents combined with the phenotypic analysis are carried out for identification of relevant region for the resistance reaction and finally for identification of new allelic variants of the resistance gene. Evaluating all described datasets should narrow down the relevant region for the resistance reaction.
A set of genotypes harboring the different RLK1 alleles have been inoculated with other plant pathogens like Southern corn leaf blight (Bipolaris maydis), Common rust (Puccinia sorghi) and Diplodia macrospora (Stenocarpella macrospora). A common feature of these pathogens is that the infection relies on a very similar biology of the fungi and on the fact that they penetrate the host via leaf tissue. A first experiment with Southern corn leaf blight (Bipolaris maydis) indicates also a resistance reaction of the HT2- and HTN-allele of RLK1.
4. Introducing Resistance to NCLB Caused by Helminthosporium turcicum into a Susceptible Genotype Via Agrobacterium-Mediated Transformation
Three different constructs with the HT2 cDNA sequence (SEQ ID NO: 2) under promoters with different activity have been transformed into the susceptible maize genotype A188: Construct A with the HT2 cDNA sequence (SEQ ID NO: 2), the native promoter region of ˜1980 bp (SEQ ID NO: 4) and terminator region (SEQ ID NO: 5) (vector p7U, see
5. Introducing Resistance to NCLB Caused by Helminthosporium turcicum into a Susceptible Genotype Via Gene Editing
The allele of RLK1 in susceptible maize genotype A188 has been identified, sequenced and a cDNA predicted (SEQ ID NO: 21). A comparison between the cDNA of this A188 allele and cDNA of HT2 shows that the sequences show a sequence identity of 99% (
| Number | Date | Country | Kind |
|---|---|---|---|
| 17187309.4 | Aug 2017 | EP | regional |
| 17206305.9 | Dec 2017 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/072665 | 8/22/2018 | WO | 00 |
