RESISTANCE GENE TO ROOT BEARNESS

Description

FIELD OF THE INVENTION

[0001] The present invention relates to a nucleic acid molecule encoding a polypeptide capable of conferring resistance to a pathogen, in particular to the "Beet Necrotic Yellow Vein Virus" (BNYVV), in a plant, in particular of the genus Beta, in which the polypeptide is expressed, as explained in more detail below. The invention further relates to a transgenic plant cell, a transgenic plant, a BNYVV-resistant plant or parts thereof, or a seed of a plant comprising the nucleic acid molecule in which resistance is conferred by introducing one or more mutations. Methods for producing a BNYVV-resistant plant, for identifying a BNYVV-resistant plant of the genus Beta, and for controlling BNYVV infestation of plants of the genus Beta are also encompassed by the present invention. BACKGROUND OF THE INVENTION

[0002] Root beard disease (rhizomania) is the most economically important sugar beet disease worldwide, causing yield losses of 50% or more. The disease, also called "root beard," is caused by the Beet Necrotic Yellow Vein Virus (BNYVV) and is caused by the soil-borne protozoan Polymyxa betae A BNYVV infection manifests itself in increased proliferation of thin roots and lateral roots and in the development of a greatly reduced root body with reduced sugar content. Infected plants exhibit reduced water uptake and are thus more sensitive to drought stress. If the infection spreads to the entire plant, yellowing of the leaf veins, necrotic lesions, and yellow spots on the leaves occur. Since a curative treatment of the disease, as with other viral diseases, is not possible, damage can only be prevented by cultivating resistant varieties. The development of genotypes with genetic resistance to rhizomania is therefore crucial for sugar beet breeding.

[0003] The first breeding programs for rhizomania resistance began in Italy in 1981 when it was recognized that satisfactory genetic variability for rhizomania resistance existed in commercial germplasm. Plants and genotypes that exhibited mitigated disease symptoms and nearly normal root weight and sugar content were selected. A multigenic resistance source called "Alba type" was successfully obtained from crosses with Beta vulgaris L. subsp. maritima (L.) to identify.

[0004] In 1982, another resistance type was developed that showed improved protection against rhizomania. This appeared two years later in the variety Rizor and was classified as a monogenic, dominant resistance. Another monogenic, dominant resistance was discovered in 1983 during variety trials in California by the breeding company "Holly Sugar Company," which was introduced in Europe just three years later (Lewellen). et al. 1987). This resistance, known as RZ-1 (also called "Holly"), became the most widely used source and quickly replaced Rizor. A few years later, a new source of rhizomania resistance was discovered in the Beta maritima Accession WB42 from Denmark was discovered. It showed a different mapping position on chromosome III than RZ-1 and also conferred an increased resistance level compared to RZ-1. This new major gene was designated RZ-2. Meanwhile, RZ-3 from the Beta maritima Accession WB41 is known, which is probably an allelic variant of RZ-2.

[0005] To date, RZ-3 is the only rhizomania resistance gene whose functional background, i.e., its genetic structure, has been elucidated (see German patent application DE 10 2013 010 026 A2), which has significantly improved the genetic use of the gene. Thus, there is a continuing desire to better characterize both known and new sources of resistance genetically in order to advance the development of molecular markers. This could further optimize elite sugar beet lines by eliminating linkage drag, but also identify new sources of resistance.

[0006] For sustainable breeding against root beard disease, which is intended to counteract the risk of resistance-breaking BNYVV isolates, it is necessary to continuously identify new resistance genes and integrate them into the gene pools of crops such as sugar beet. This object is achieved according to the invention by the embodiments characterized in the claims and the description. SUMMARY OF THE INVENTION

[0007] The present invention relates to a nucleic acid molecule capable of conferring resistance to a pathogen, in particular to the "Beet Necrotic Yellow Vein Virus" (BNYVV), in a plant, in particular of the genus Beta, in which a polypeptide encoded by the nucleic acid molecule is expressed. The invention further relates to a transgenic plant cell, a transgenic plant, a BNYVV-resistant plant or parts thereof, or a seed of a plant comprising the nucleic acid molecule, in which the resistance is conferred by the introduction of one or more mutations. Methods for producing a BNYVV-resistant plant, for identifying a BNYVV-resistant plant of the genus Beta, and for controlling the infestation of plants of the genus Beta with BNYVV are also encompassed by the present invention.

[0008] The present invention is defined by the features of the independent claims.

[0009] According to a first aspect, the present invention relates to a nucleic acid molecule encoding a polypeptide capable of conferring resistance to a pathogen in a plant in which the polypeptide is expressed, characterized in that the nucleic acid molecule comprises a nucleotide sequence selected from (a) a nucleotide sequence encoding a polypeptide having an amino acid sequence which is at least 80% identical to SEQ ID NO: 2, wherein the nucleic acid molecule has one or more mutations which result in the polypeptide encoded by the nucleic acid molecule having an amino acid different from lysine (K) at the position corresponding to position 307 in the reference amino acid sequence SEQ ID NO: 2 and / or having an amino acid different from glutamine (Q) at the position corresponding to position 437 in the reference amino acid sequence SEQ ID NO: 2;and (b) a nucleotide sequence which hybridizes with the complementary sequence to SEQ ID NO: 1 under stringent conditions, wherein the nucleotide sequence has one or more of the following nucleotide substitutions at those positions corresponding to the positions in the reference nucleotide sequence SEQ ID NO: 1: C instead of A at position 919; G instead of A at position 1310; A instead of G at position 1697; A instead of C at position 2191; and / or T instead of C at position 2491; ; preferably wherein the pathogen is Beet necrotic yellow vein virus (BNYVV) and the plant is a plant of the genus Beta, preferably sugar beet (Beta vulgaris L.).

[0010] Said nucleic acid molecule can be characterized in that the encoded polypeptide further comprises (i) at the position corresponding to position 566 in the reference amino acid sequence SEQ ID NO: 2, an amino acid different from arginine (R), and / or (ii) at the position corresponding to position 731 in the reference amino acid sequence SEQ ID NO: 2, an amino acid different from glutamine (Q), and / or (iii) at the position corresponding to position 831 in the reference amino acid sequence SEQ ID NO: 2, an amino acid different from proline (P).

[0011] The said nucleic acid molecule can be characterized in that the encoded polypeptide has (i) a glutamine (Q) at the position corresponding to position 307 in the reference amino acid sequence SEQ ID NO: 2, and / or (ii) an arginine (R) at the position corresponding to position 437 in the reference amino acid sequence SEQ ID NO: 2, and / or (iii) a histidine (H) at the position corresponding to position 566 in the reference amino acid sequence SEQ ID NO: 2, and / or (iv) a lysine (K) at the position corresponding to position 731 in the reference amino acid sequence SEQ ID NO: 2, and / or (v) a serine (S) at the position corresponding to position 831 in the reference amino acid sequence SEQ ID NO: 2.

[0012] The nucleic acid molecule in question may be characterized in that the nucleic acid molecule has one or more of the following nucleotide substitutions at those positions corresponding to the positions in the reference nucleotide sequence SEQ ID NO: 1: (a) C instead of A at position 919; (b) G instead of A at position 1310; (c) A instead of G at position 1697; (d) A instead of C at position 2191; and / or (e) T instead of C at position 2491.

[0013] Said nucleic acid molecule can be characterized in that the nucleic acid molecule encodes a polypeptide having an amino acid sequence according to SEQ ID NO: 4 and / or comprises the coding DNA sequence according to SEQ ID NO: 3.

[0014] According to a second aspect, the present invention relates to a vector comprising the above-mentioned nucleic acid molecule.

[0015] According to a third aspect, the present invention relates to a transgenic plant cell, preferably from a plant of the genus Beta, which comprises the above-mentioned nucleic acid molecule as a transgene, optionally under the control of a heterologous promoter, or the above-mentioned vector.

[0016] According to a fourth aspect, the present invention relates to a transgenic plant, preferably of the genus Beta, or a part thereof, comprising a plant cell.

[0017] According to a fifth aspect, the present invention relates to a BNYVV-resistant plant of the species Beta vulgaris or a part thereof, in which said nucleic acid molecule is endogenously present, or in which the one or more mutations as defined above have been introduced into an endogenous nucleic acid molecule having a nucleotide sequence comprising the sequence according to SEQ ID NO: 1 or which hybridizes with the complementary sequence to SEQ ID NO: 1 under stringent conditions, wherein the plant or a part thereof does not belong to the subspecies Beta vulgaris subsp. maritima heard.

[0018] The above-mentioned plant is a hybrid plant or double haploid plant, and / or the nucleic acid molecule or the one or more mutations are heterozygous or homozygous.

[0019] According to a sixth aspect, the present invention relates to a seed from the above-mentioned plant, wherein the seed comprises one or more of the aforementioned nucleic acid molecules or vectors transgenically or endogenously.

[0020] According to a seventh aspect, the present invention relates to a method for producing a BNYVV-resistant plant, comprising the following steps: (a) mutagenizing plant cells and subsequently regenerating plants from the mutagenized plant cells or mutagenizing plants, and (b) identifying a plant from (a) which has the one or more mutations in an endogenous nucleic acid molecule.

[0021] According to an eighth aspect, the present invention relates to a method for identifying a plant of the genus Beta which is resistant to the pathogen BNYVV, characterized in that the method comprises the following step: (i) detecting the presence of said nucleic acid molecule, wherein the polypeptide encoded thereby has (i) a glutamine (Q) at the position corresponding to position 307 in the reference amino acid sequence SEQ ID NO: 2, or (ii) an arginine (R) at the position corresponding to position 437 in the reference amino acid sequence SEQ ID NO: 2, and / or the expression of the nucleic acid molecule, wherein the nucleic acid molecule has the following nucleotide substitutions at the positions corresponding to the positions in the reference nucleotide sequence SEQ ID NO: 1: (a) C instead of A at position 919, or (b) G instead of A at position 1310; in the plant ora sample thereof; and / or (ii) detecting the presence of said nucleic acid molecule, wherein the polypeptide encoded thereby has (i) a glutamine (Q) at the position corresponding to position 307 in the reference amino acid sequence SEQ ID NO: 2, and (ii) an arginine (R) at the position corresponding to position 437 in the reference amino acid sequence SEQ ID NO: 2, and / or the expression of said nucleic acid molecule, wherein the nucleic acid molecule has the following nucleotide substitutions at the positions corresponding to the positions in the reference nucleotide sequence SEQ ID NO: 1: (a) C instead of A at position 919, and (b) G instead of A at position 1310; in the plant or a sample thereof, and optionally (iii) selecting the BNYVV-resistant plant.

[0022] According to a ninth aspect, the present invention relates to a population of plants comprising the above-mentioned plants or plants which have been identified and optionally selected by the above-mentioned method.

[0023] According to a tenth aspect, the present invention relates to a method for controlling infestation with the pathogen Beet Necrotic Yellow Vein Virus (BNYVV) in the agricultural or horticultural cultivation of plants of the genus Beta, comprising I) identifying and selecting plants of the genus Beta using the above method and II) growing the plants from I) or progeny thereof.

[0024] First, some of the terms used in this application are explained in more detail below: The term "about" in connection with the specification of a length of a nucleotide sequence means a deviation of + / - 10,000 base pairs, + / - 5,000 base pairs or + / - 1,000 base pairs, preferably by + / - 200 base pairs or + / - 100 base pairs and particularly preferably by + / - 50 base pairs.

[0025] A "plant of the genus Beta" belongs to the Amaranthaceae family. These plants include plants of the species Beta macrocarpa , Beta vulgaris, Beta lomatogona, Beta macrorhiza , Beta corolliflora , Beta trigyna and Beta nana. A plant of the species Beta vulgaris is in particular a plant of the subspecies Beta vulgaris subsp. vulgaris. These include, for example, Beta vulgaris subsp. vulgaris var. altissima (sugar beet in the strict sense), Beta vulgaris ssp. vulgaris var. vulgaris (chard), Beta vulgaris ssp. vulgaris var. conditiva (Beetroot), Beta vulgaris ssp. vulgaris var. crassa / alba (fodder beet).

[0026] "Hybridization" or "hybridization" is understood to mean a process in which a single-stranded nucleic acid molecule binds to a largely complementary nucleic acid strand, i.e., forms base pairs with it. Standard hybridization procedures are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001. Preferably, this means 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 base pair with the largely complementary nucleic acid strand. The possibility of such binding depends on the stringency of the hybridization conditions. The term "stringency" refers to the hybridization conditions.High stringency exists when base pairing is difficult, low stringency 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, stringency can be increased by increasing the temperature and / or decreasing the salt content. "Stringent hybridization conditions" are those conditions under which hybridization occurs predominantly only between homologous nucleic acid molecules. The term "hybridization conditions" refers not only to the conditions prevailing during the actual annealing of the nucleic acids, but also to those prevailing during the subsequent washing steps.Stringent hybridization conditions are, for example, conditions under which predominantly only those nucleic acid molecules hybridize that exhibit at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity. Stringent hybridization conditions include, for example, hybridization in 4 x SSC at 65°C followed by multiple washings in 0.1 x SSC at 65°C for a total of approximately 1 hour. The term "stringent hybridization conditions" used here can also mean hybridization at 68°C in 0.25 M sodium phosphate, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA for 16 hours followed by two washings with 2 x SSC and 0.1% SDS at 68°C. Hybridization preferably takes place under stringent conditions.

[0027] "Complementary" nucleotide sequence, with reference to a nucleic acid in the form of double-stranded DNA, means that the second DNA strand, complementary to the first DNA strand, has the nucleotides corresponding to the bases of the first strand according to the base pairing rules.

[0028] An "isolated nucleic acid molecule" is a nucleic acid molecule that has been removed from its natural or original environment. The term also includes a synthetically produced nucleic acid molecule. An "isolated polypeptide" is a polypeptide that has been removed from its natural or original environment. The term also includes a synthetically produced polypeptide.

[0029] A "molecular marker" is a nucleic acid that is polymorphic in a plant population and is used as a reference or landmark. A marker for detecting a recombination event should be suitable for monitoring differences or polymorphisms within a plant population. Thus, such a marker is capable of detecting and distinguishing different allelic states (alleles). The term "molecular marker" also refers to nucleotide sequences that are complementary or at least largely complementary or homologous to genomic sequences, for example, nucleic acids used as probes or primers. For markers, these differences are found at the DNA level and are, for example, polynucleotide sequence differences such as SSRs ( simple sequence repeats ), RFLPs ( restriction fragment length polymorphisms ), FLPs ( fragment length polymorphisms ) or SNPs ( single nucleotide polymorphisms ) .The markers can be derived from genomic or expressed nucleic acids such as spliced RNA, cDNA, or ESTs, and can also refer to nucleic acids used as probes or primer pairs, which are suitable for amplifying a sequence fragment using PCR-based methods. Markers describing genetic polymorphisms (between parts of a population) can be detected using well-established state-of-the-art methods (An Introduction to Genetic Analysis, 7th Edition, Griffiths, Miller, Suzuki et al., 2000). These include, for example, DNA sequencing, PCR-based sequence-specific amplification, detection of RFLPs, detection of polynucleotide polymorphisms using allele-specific hybridization (ASH), detection of amplified variable sequences of the plant genome, detection of a 3SR ( self-sustained sequence replication ), detection of SSRs, SNPs, RFLPs or AFLPs ( amplified fragment length polymorphisms ) .Furthermore, the methods for detecting ESTs ( expressed sequence tags ) and SSR markers derived from EST sequences and RAPD ( randomly amplified polymorphie DNA ). Depending on the context, the term marker in the description can also refer to a specific chromosome position in the genome of a species where a specific marker (e.g., SNP) can be found.

[0030] A "promoter" is a non-translated regulatory DNA sequence, typically upstream of a coding region, which contains the binding site for RNA polymerase and initiates transcription of the DNA. A promoter also contains other elements that act as regulators of gene expression (e.g., cis-regulatory elements). A "core or minimal promoter" is a promoter that contains at least the basic elements needed for transcription initiation (e.g., a TATA box and / or initiator).

[0031] A "pathogen" is an organism that, through interaction with a plant, causes disease symptoms in one or more organs of the plant. These pathogens include, for example, animal, fungal, bacterial, or viral organisms, or oomycetes.

[0032] A "pathogen infection" is defined as the earliest point in time at which a pathogen interacts with a plant host tissue. For example, in the case of the viral pathogen BNYVV, this is caused by the protozoan Polymyxa betae transmitted. Polymyxa forms spores that can persist in the soil for many decades. The virus also survives in these spores. When these persistent spores germinate into mobile zoospores, the virus can enter cells of the plant host tissue and interact with the host there (Esser (2000) Cryptogams 1: Cyanobacteria, Algae, Fungi, Lichens, Practical and Textbook. Springer-Verlag, Berlin, Heidelberg, 3rd edition).

[0033] Plant "organs" include, for example, leaves, stems, roots, hypocotyls, vegetative buds, meristems, embryos, anthers, ovules, seeds, or fruits. "Plant parts" or "plant part(s)" include, but are not limited to, the stem, leaves, flowers, inflorescences, roots, fruits, seeds, and pollen. The term "plant parts" or "plant part(s)" also refers to a combination of several organs, such as a flower or a seed, or a part of an organ, such as a cross-section through the stem. Plant "tissue" includes, for example, callus tissue, storage tissue, meristematic tissue, leaf tissue, shoot tissue, root tissue, plant tumor tissue, or reproductive tissue, as well as the formative tissue, ground tissue (the so-called parenchyma), conductive tissue, strengthening tissue, and the covering tissue (the so-called epidermis). However, this list does not limit the fabric.Plant "cells" include, for example, isolated cells with a cell wall or aggregates thereof or protoplasts.

[0034] In the context of the present invention, the term "regulatory sequence" refers to a nucleotide sequence that influences specificity and / or expression levels, for example, by mediating a particular tissue specificity. Such a regulatory sequence can be located upstream of the transcription initiation point of a minimal promoter, but also downstream of it, such as in a transcribed but untranslated leader sequence or within an intron.

[0035] The term "resistance" is to be understood broadly and covers the range of protection from delaying to completely inhibiting the development of the disease. An example of an important pathogen is the Beet Necrotic Yellow Vein Virus (BNYVV). Preferably, a resistant plant cell of the invention or a resistant plant of the invention achieves resistance to BNYVV. Resistance to a pathogen is equivalent to resistance to the disease caused by that pathogen; for example, resistance to BNYVV also means resistance to rhizomania.

[0036] "Transgenic plant" refers to a plant in whose genome at least one polynucleotide is integrated. This can be a heterologous polynucleotide. Preferably, the polynucleotide is stably integrated, meaning that the integrated polynucleotide is stably maintained in the plant, expressed, and can also be stably inherited by the offspring. The stable introduction of a polynucleotide into the genome of a plant also includes integration into the genome of a plant of the previous parental generation, whereby the polynucleotide can be stably inherited.The term "heterologous" means that the introduced polynucleotide originates, for example, from a cell or organism with a different genetic background within the same species or a different species, or is homologous to the prokaryotic or eukaryotic host cell but is located in a different genetic environment, thus differing from any naturally occurring corresponding polynucleotide. A heterologous polynucleotide can be present in addition to a corresponding endogenous gene.

[0037] Embodiments and aspects of the present invention will be described by way of example with reference to the accompanying drawings and figures. Abb. 1: (A) Genomic DNA sequence of the root beard (WB) sensitive allele wb -s of the resistance gene wb (SEQ ID NO: 1) from an ss genotype identified as NBS-LRR (NB-ARC domain gene) by fine mapping. Analysis of the progeny from the four closest recombinant plants (two direct recombinants on the left and two direct recombinants on the right surrounding the gene) has narrowed down the NBS-LRR gene to a single gene. The sequence shown includes the coding region of the predicted protein sequence ( B, SEQ ID NO: 2) of the resistance protein WB-s. (C) Genomic DNA sequence of a WB resistant allele wb -R of the resistance gene wb (SEQ ID NO: 3) encompassing the coding region of the predicted protein sequence. Diagnostic polymorphisms resulting in amino acid substitutions are underlined and highlighted in bold. (D) Amino acid sequence of a WB resistant allele from the genomic DNA sequence wb- R of the resistance gene wb (SEQ ID NO: 3) encoded protein. Diagnostic polymorphisms are underlined and highlighted in bold. Experiments within the scope of the present invention, including sequence analyses of the WB-resistant genotypes versus of the predominant sensitive genotype and the amino acid sequence contained in the genome database revealed that the genomic DNA sequence of the WB resistant wb -R alleles, the correlating NBS-LRR gene with the reading frame shown in SEQ ID NO: 3 has one or more amino acid substitutions, so the observed resistance is due to mutations in this gene. Based on the analysis of the recombinants to date, one of the two amino acid substitutions, K307Q and / or Q437R, can be considered causal for the resistance. DETAILED DESCRIPTION OF THE INVENTION

[0038] The present invention relates to a nucleic acid molecule capable of conferring resistance to a pathogen, in particular to the "Beet Necrotic Yellow Vein Virus" (BNYVV), in a plant, in particular of the genus Beta, in which the polypeptide encoded by the nucleic acid molecule is expressed. The present invention is based on the genetic fine mapping, identification, isolation, and characterization of a gene originally derived from the donor Beta vulgaris subsp. maritima whose presence in a plant, in particular in sugar beet, correlates with or is causative of the plant's resistance to root beard (WB), and whose nucleotide and encoded amino acid sequence differs by at least one nucleotide or amino acid exchange compared to the Figur 1A und B shown nucleotide and amino acid sequence of the identified NBS-LRR gene.

[0039] According to the first aspect, the present invention relates to a nucleic acid molecule encoding a polypeptide capable of conferring resistance to a pathogen in a plant in which the polypeptide is expressed, characterized in that the nucleic acid molecule comprises a nucleotide sequence selected from (a) a nucleotide sequence encoding a polypeptide having an amino acid sequence which is at least 80% identical to SEQ ID NO: 2, wherein the nucleic acid molecule has one or more mutations which result in the polypeptide encoded by the nucleic acid molecule having an amino acid different from lysine (K) at the position corresponding to position 307 in the reference amino acid sequence SEQ ID NO: 2 and / or having an amino acid different from glutamine (Q) at the position corresponding to position 437 in the reference amino acid sequence SEQ ID NO: 2;and (b) a nucleotide sequence which hybridizes with the complementary sequence to SEQ ID NO: 1 under stringent conditions, wherein the nucleotide sequence has one or more of the following nucleotide substitutions at those positions corresponding to the positions in the reference nucleotide sequence SEQ ID NO: 1: C instead of A at position 919; G instead of A at position 1310; A instead of G at position 1697; A instead of C at position 2191; and / or T instead of C at position 2491; ; preferably wherein the pathogen is Beet necrotic yellow vein virus (BNYVV) and the plant is a plant of the genus Beta, preferably sugar beet (Beta vulgaris L.).

[0040] The nucleic acid molecule can be an isolated nucleic acid molecule. Preferably, it is DNA, and particularly preferably cDNA or coding DNA. Preferably, the polypeptide encoded by the nucleic acid molecule according to the invention confers resistance to the viral pathogen. "Beet Necrotic Yellow Vein Virus" (BNYVV), which causes the plant disease rhizomania and is caused by the soil-borne protozoa Polymyxa betae Furthermore, the polypeptide encoded by the nucleic acid molecule according to the invention confers resistance to this pathogen, particularly in a plant of the genus Beta. Preferably, the plant is a plant of the species Beta vulgaris, particularly preferably a plant of the subspecies Beta vulgaris subsp. vulgaris These include, for example, the cultivated species sugar beet, beetroot, fodder beet, leaf chard and stem chard.

[0041] In one embodiment of the present invention, the nucleic acid molecule according to the invention comprises a nucleotide sequence encoding a polypeptide having an amino acid sequence that is at least 80% identical to SEQ ID NO: 2, wherein the nucleic acid molecule has one or more mutations that result in the polypeptide encoded by the nucleic acid molecule having an amino acid different from lysine (K) at the position corresponding to position 307 in the reference amino acid sequence SEQ ID NO: 2 and / or having an amino acid different from glutamine (Q) at the position corresponding to position 437 in the reference amino acid sequence SEQ ID NO: 2. One or both of the two or both of the mentioned mutations in the amino acid sequence have been putatively identified as causal for resistance to rhizomania.

[0042] As explained in the examples and the figure legend, the identified gene is a resistance gene / protein of the NBS-LRR type, which is characterized by specific structural motifs. The general structure of such resistance proteins in plants has already been well studied (Martin et al., Annual Review Plant Biology 54 (2003), 23-61). However, the principle of the structural design, particularly of the so-called LRR domain, which is considered a potential recognition domain for mostly unknown pathogenic effectors, is unpredictable, and in general, the functional background of the resistance genes, i.e., the genetic structure, is largely unknown. Consequently, the identification of a BNYVV resistance-mediating gene or protein based solely on the known structural motifs is impossible.Furthermore, the sequence regions around these resistance genes are often repetitive, which makes the development of diagnostic markers and the assembly of sequences particularly difficult.

[0043] The identified resistance protein belongs to the NBS-LRR type and has at least one leucine-rich domain (LRR) corresponding to amino acid positions 558 to 594 of SEQ ID NO: 4, preferably amino acid positions 542 to 594 of SEQ ID NO: 4, amino acid positions 604 to 634 of SEQ ID NO: 4, preferably amino acid positions 582 to 634 of SEQ ID NO: 4, amino acid positions 760 to 790 of SEQ ID NO: 4 or amino acid positions 838 to 869 of SEQ ID NO: 4, and / or at least one AAA ATPase domain corresponding to amino acid positions 177 to 289 of SEQ ID NO: 4 or amino acid positions 156 to 263 of SEQ ID NO: 4. The identified ATPase domain is most likely the NB-ARC domain, which has a central nucleotide-binding function. Without being bound to any particular theory, this functional ATPase domain is likely to regulate the activity of the resistance protein.

[0044] In a further embodiment, the nucleic acid molecule according to the invention comprises a nucleotide sequence which hybridizes with the complementary sequence to SEQ ID NO: 1 under stringent conditions, wherein one or more nucleotides are exchanged at positions 919-921 and / or 1309-1311, resulting in an amino acid exchange.

[0045] These amino acid or nucleotide exchanges can be carried out using conventional methods known in the art, for example by site-directed mutagenesis, PCR-mediated mutagenesis, transposon mutagenesis, TILLING, genome engineering, for example by means of meganucleases, zinc finger nucleases, TALENS and CRISPR / Cas, etc. Furthermore, further substitutions, deletions, insertions, additions and / or any other changes can be introduced into the nucleotide sequence, either alone or in combination, which change the nucleotide sequence but fulfill the same function as the starting sequence, here the nucleotide sequence according to the invention, which encodes a polypeptide with the amino acid sequence according to the invention, which confers resistance to root beard disease (rhizomania).Therefore, in a further embodiment, the invention encompasses a nucleotide sequence encoding a polypeptide that is a derivative of the polypeptide encoded by the nucleotide sequence of the invention or that comprises the amino acid sequence of the invention. A derivative of the polypeptide represents a derived amino acid sequence that has at least one substitution, deletion, insertion, or addition of one or more amino acids, while retaining the functionality of the encoded polypeptide / protein.

[0046] The substitution of one amino acid with another with the same, equivalent, or similar chemical-physical properties is referred to as a "conservative exchange" or "semiconservative exchange." Examples of physical-chemical properties of an amino acid include hydrophobicity or charge. The skilled person knows which amino acid substitutions constitute conservative or semiconservative exchanges. Furthermore, general technical knowledge allows the skilled person to recognize, identify, and demonstrate which amino acid deletions and additions are harmless to the functionality of the resistance protein and at which positions these are possible.The person skilled in the art is aware that, in the case of the present NBS-LRR protein, modifications of the amino acid sequence (substitutions, deletions, insertions or additions of one or more amino acids) must in particular preserve the functionality of the conserved domains defined above and that, therefore, only limited modifications of the above are possible in these domains.

[0047] Thus, the invention encompasses a functional fragment of the nucleotide sequence according to the invention. The term "fragment" encompasses genes with a nucleotide sequence sufficiently similar to the above-mentioned nucleotide sequence. The term "sufficiently similar" means a first nucleotide sequence or amino acid sequence that has a sufficient or minimal number of identical or equivalent nucleotides or amino acid residues relative to a second nucleotide or amino acid sequence.

[0048] With regard to the amino acid sequence, this has a common structural domain and / or has common functional activity even after modification by an above-mentioned method. Amino acid sequences that have an identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the nucleotide sequence or amino acid sequence according to the invention are defined here as sufficiently similar. Preferably, such nucleotide sequences which encode a derivative or a derived amino acid sequence can be generated either directly or indirectly (for example via amplification or replication steps) from a starting nucleotide sequence which covers the entire length or at least part of the nucleotide sequence according to the invention.

[0049] Accordingly, the present invention comprises a nucleotide sequence capable of hybridizing under stringent conditions to a nucleotide sequence complementary to a nucleotide sequence according to the invention or to the nucleotide sequence encoding the amino acid sequence according to the invention.

[0050] In a further embodiment, the nucleic acid molecule according to the invention comprises a nucleotide sequence, wherein the nucleic acid molecule has one or more mutations which result in the polypeptide encoded by the nucleic acid molecule having an amino acid different from lysine (K) at the position corresponding to position 307 in the reference amino acid sequence SEQ ID NO: 2, and / or having an amino acid different from glutamine (Q) at the position corresponding to position 437 in the reference amino acid sequence SEQ ID NO: 2, and having an amino acid different from arginine (R) at the position corresponding to position 566 in the reference amino acid sequence SEQ ID NO: 2, and / or (ii) having an amino acid different from glutamine (Q) at the position corresponding to position 731 in the reference amino acid sequence SEQ ID NO: 2,and / or (iii) has an amino acid other than proline (P) at the position corresponding to position 831 in the reference amino acid sequence SEQ ID NO: 2. The first two substitutions were identified as responsible for the resistance, as mentioned above. Although the role of the last three substitutions in conferring resistance is still unclear, they are diagnostic and can thus be advantageously used in detection and / or selection methods.

[0051] In a further embodiment, the nucleic acid molecule according to the invention comprises a nucleotide sequence, wherein the encoded polypeptide has a glutamine (Q) at the position which corresponds to position 307 in the reference amino acid sequence SEQ ID NO: 2, and / or has an arginine (R) at the position which corresponds to position 437 in the reference amino acid sequence SEQ ID NO: 2, and / or has a histidine (H) at the position which corresponds to position 566 in the reference amino acid sequence SEQ ID NO: 2, and / or has a lysine (K) at the position which corresponds to position 731 in the reference amino acid sequence SEQ ID NO: 2, and / or has a serine (S) at the position which corresponds to position 831 in the reference amino acid sequence SEQ ID NO: 2.These amino acid substitutions preferably occur through the substitution of one or more nucleotides in the nucleic acid molecule of SEQ ID NO: 1, wherein preferably one nucleotide is substituted by another. Accordingly, the nucleic acid molecule according to the invention is characterized in that the nucleic acid molecule has one or more of the following nucleotide substitutions at those positions which correspond to the positions in the reference nucleotide sequence SEQ ID NO: 1: C instead of A at position 919; G instead of A at position 1310; A instead of G at position 1697; A instead of C at position 2191; and / or T instead of C at position 2491. In a preferred embodiment, the nucleic acid molecule according to the invention is characterized in that it encodes a polypeptide with an amino acid sequence according to SEQ ID NO: 4 and / or comprises the coding DNA sequence according to SEQ ID NO: 3; see also. Abbildung 1 and the figure legend as well as the examples.

[0052] Furthermore, the present application discloses a recombinant DNA molecule comprising the sequences of said nucleic acid molecule. Preferably, the recombinant DNA molecule further comprises or is associated with / operably linked to a regulatory sequence, preferably with a promoter sequence and / or other sequences of transcription or translation control elements, wherein the regulatory sequence controlling the expression of a gene comprising the nucleic acid molecule according to the invention is characterized in that the regulatory sequence is capable of mediating or modulating the expression of a heterologous DNA sequence as a result of a pathogen infection. Preferably, the heterologous DNA sequence is a nucleotide sequence encoding a component of plant pathogen defense (e.g.,: resistance genes (R genes) or genes which code for enzymes involved in signal transfer such as kinases or phosphatases as well as for G protein) or which code for a pathogenic effector (so-called avirulence genes (. avr )) .

[0053] The present invention further discloses a polypeptide encoded by the nucleic acid molecule according to the invention and a functional and / or immunologically active fragment thereof, as well as an antibody that specifically binds to the polypeptide or to its fragment. The polypeptide particularly preferably has an amino acid sequence according to SEQ ID NO: 4. The recombinant production of proteins, polypeptides and fragments is familiar to the person skilled in the art and is described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001 or Wingfield, PT 2008. Production of Recombinant Proteins. Current Protocols in Protein Science. 52:5.0:5.0.1-5.0.4. Polyclonal or monoclonal antibodies to the protein of the present invention can be produced by the person skilled in the art using known methods, as described in E. Harlow et al., Editors Antibodies: A Laboratory Manual (1988). The production of monoclonal antibodies as well as Fab and F(ab') 2 fragments, which are also useful in protein detection methods, can be carried out by various common methods as described in Goding, Mononoclonal Antibodies: Principles and Practice, pp. 98-118, New York: Academic Press (1983). The antibody can then be used to screen expression cDNA libraries to identify identical, homologous, or heterologous genes by immunological screening (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 or Ausubel et al., 1994, "Current Protocols in Molecular Biology", John Wiley & Sons). In particular, the present invention relates to antibodies which recognize a gene produced by the invention. wb -R allele and essentially not the sensitive wb -s allele encoded polypeptides, ie at least by a factor of 2, preferably by a factor of 5, and more preferably by a factor of 10 or more lower than that encoded by the wb -R allele encoded polypeptide.

[0054] The invention further relates to vectors comprising the nucleic acid molecule or the recombinant DNA molecule according to the invention. The vector can be a plasmid, a cosmid, a phage, or an expression vector, a transformation vector, a shuttle vector, or a cloning vector. It can be double- or single-stranded, linear or circular, or can transform a prokaryotic or eukaryotic host either by integration into its genome or extrachromosomally. Preferably, the nucleic acid molecule or DNA molecule according to the invention is operatively linked in an expression vector to one or more regulatory sequences that allow transcription and, optionally, expression in a prokaryotic or eukaryotic host cell; see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001.These regulatory sequences are preferably promoters or terminators, in particular a transcription initiation start point, a ribosome binding site, an RNA-processing signal, a transcription termination site, and / or a polyadenylation signal. For example, the nucleic acid molecule is under the control of a suitable promoter and / or a terminator. Suitable promoters can be promoters that are constitutively induced (e.g., the 35S promoter from the "Cauliflower mosaic virus" (Odell et al., Nature 313 (1985), 810-812). Particularly suitable are promoters that are pathogen-inducible (e.g., the PR1 promoter from parsley (Rushton et al., EMBO J. 15 (1996), 5690-5700). Particularly suitable pathogen-inducible promoters are synthetic orChimeric promoters, which do not occur naturally, are composed of multiple elements and contain a minimal promoter and at least one cis-regulatory element upstream of the minimal promoter, which serves as a binding site for specific transcription factors. Chimeric promoters are designed according to the desired requirements and are induced or repressed by different factors. Examples of such promoters can be found in WO 00 / 29592, WO 2007 / 147395, and WO 2013 / 091612. A suitable terminator is, for example, the nos terminator (Depicker et al., J. Mol. Appl. Genet. 1 (1982), 561-573). The vectors usually also contain indicator / reporter genes or resistance genes to detect the transfer of the desired vector or DNA molecule / nucleic acid molecule and to select the individuals containing them, since direct detection via gene expression is usually rather difficult.Since the nucleic acid molecule according to the invention itself encodes a polypeptide which, with the aforementioned mutations, represents the protein that confers resistance to rhizomania, it is not essential for expression in plant cells to provide another resistance gene, but is preferably provided to allow rapid selection.

[0055] Examples of indicator / reporter genes include the luciferase gene and the gene encoding green fluorescent protein (GFP). These also allow studies on the activity and / or regulation of a gene's promoter. Examples of resistance genes, especially for plant transformation, are the neomycin phosphotransferase gene, the hygromycin phosphotransferase gene, or the gene encoding phosphinothricin acetyltransferase. However, this does not exclude other indicator / reporter genes or resistance genes known to the person skilled in the art. In a preferred embodiment, the vector is a plant vector.

[0056] Furthermore, the present application discloses host cells comprising the vectors, recombinant DNA molecules and / or nucleic acid molecules according to the invention. A host cell within the meaning of the invention can be a prokaryotic (e.g., bacterial) or eukaryotic cell (e.g., a plant cell or a yeast cell). Preferably, the host cell is an Agrobacterium such as Agrobacterium tumefaciens or Agrobacterium rhizogenes or a plant cell.

[0057] The person skilled in the art is aware of numerous methods such as conjugation or electroporation with which he can introduce the nucleic acid molecule according to the invention, the recombinant DNA molecule and / or the vector of the present invention into an Agrobacterium, as well as methods such as various transformation methods (biolistic transformation, Agrobacterium-mediated transformation) with which he can introduce the nucleic acid molecule according to the invention, the DNA molecule and / or the vector of the present invention into a plant cell (Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001).

[0058] The present invention further preferably relates to a transgenic plant cell comprising the nucleic acid molecule or DNA molecule according to the invention as a transgene or the vector of the present invention. Such a transgenic plant cell is, for example, a plant cell that has been transformed, preferably stably, with the nucleic acid molecule, DNA molecule according to the invention, or with the vector of the present invention. In a preferred embodiment of the transgenic plant cell, the nucleic acid molecule is operatively linked to one or more regulatory sequences that allow transcription and, optionally, expression in the plant cell. The overall construct comprising the nucleic acid molecule according to the invention and the regulatory sequence(s) then represents the transgene. Such regulatory sequences are, for example, a promoter, an enhancer, or a terminator.The person skilled in the art is aware of numerous functional promoters, enhancers and terminators that can be used in plants.

[0059] Preferably, a transgenic plant cell of the present invention, in particular a cell of a plant of the genus Beta, exhibits a higher resistance to a pathogen, in particular BNYVV, than a corresponding non-transformed plant cell (the plant cell without the transgene). The level of resistance, for example, to BNYVV, can be qualitatively determined in plants of the genus Beta by determining rating scores (rating schemes for plants of the genus Beta are known from the prior art, for example for sugar beet; see Mechelke (1997) Problems in Rizomaniaresistenzzuchtung, Gespräche für Pflanzenzuchtung, Resistenzzuchtung bei Zuckerrüben, Gesellschaft für Pflanzenzuchtung eV, 113-123). Higher resistance is reflected in an improvement in resistance by at least one rating score, by at least two rating scores, by at least three or more rating scores.Furthermore, the present application also discloses a method for producing a transgenic plant cell of the present invention, which comprises a step of introducing the nucleic acid molecule, DNA molecule, or vector of the present invention into a plant cell. For example, the introduction can take place by transformation, preferably by stable transformation. Suitable techniques for introduction, such as biolistic transformation, Agrobacterium-mediated transformation, or electroporation, are known to the person skilled in the art (Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001).

[0060] In a further aspect, the present invention relates to a transgenic plant and parts thereof, comprising a transgenic plant cell as described above. A part can be a cell, a tissue, an organ or a combination of several cells, tissues or organs. A combination of several organs is, for example, a flower or a seed. In a particular embodiment, the invention relates to a seed from the transgenic plant, wherein the seed comprises the nucleic acid molecule according to the invention as a transgene. Preferably, a transgenic plant of the present invention, in particular a plant of the genus Beta, shows a higher resistance to a pathogen, in particular BNYVV, than a corresponding non-transformed plant (plant without the transgene).The level of resistance, for example, to BNYVV, can be qualitatively determined in plants of the genus Beta by determining rating scores (rating schemes for plants of the genus Beta are known from the prior art, for example for sugar beet, see Mechelke (1997) Problems in Rizomania Resistance Breeding, Lectures on Plant Breeding, Resistance Breeding in Sugar Beets, Society for Plant Breeding eV, 113-123). Higher resistance is indicated by an improvement in resistance by at least one rating score, by at least two rating scores, or by at least three or more rating scores. Furthermore, the invention provides a method for producing a transgenic plant, which comprises a step of introducing the nucleic acid molecule according to the invention or the vector of the present invention into a plant cell and optionally a step of selecting a transgenic plant cell.Furthermore, such a method for producing a transgenic plant is characterized by a subsequent cut, which includes the regeneration of the transgenic plant from the transgenic plant cell generated in the first step. Methods for regeneration are known to the person skilled in the art.

[0061] In a further aspect, the present invention also relates to a method for conferring or increasing resistance to a pathogen, in particular BNYVV, in a plant, preferably a plant of the genus Beta, which comprises a step of transforming a plant cell with the nucleic acid molecule according to the invention or the vector of the present invention. Alternatively, as described above, a WB-resistant genotype can be produced from a WB-sensitive genotype by randomly or deliberately mutagenizing the existing endogenous wb-s gene.

[0062] For example, the wb gene can be modified by gene mutation using TALE nucleases (TALENs) or zinc finger nucleases (ZFNs) as well as CRISPR / Cas systems, which are described, for example, in WO 2014 / 144155 A1 (Engineering plant genomes using CRISPR / Cas systems) and in Osakabe & Osakabe, Plant Cell Physiol, 56 (2015), 389-400. This can also be achieved by using the TILLING ( Targeted Induced Local Lesions in Genomes ), whereby, as described, for example, in the German patent application DE 10 2013 101 617, point mutations are induced in the wild-type gene and then plants are selected that have a suitable, i.e., resistance-conferring mutation, such as a barley resistant to yellow mosaic virus; see DE 10 2013 101617 on pages 4, 8 and 12 in paragraphs

[0014] ,

[0026] and

[0038] . The TILLING method is also described in detail in the publication by Henikoff et al. described (Henikoff et al., Plant Physiol. 135, 2004, 630-636).

[0063] This method preferably leads to an improvement in resistance by at least one rating grade, particularly preferably to an improvement in resistance by at least two, three, or more rating grades. Rating schemes for plants of the genus Beta are known from the prior art, for example for sugar beet (Mechelke 1997). After mutagenizing the plant cells and subsequently regenerating plants from the mutagenized plant cells or mutagenizing plants, the plants can then be identified which have one or more mutations as defined above in an endogenous nucleic acid molecule, preferably with the nucleotide sequence that hybridizes with the complementary sequence to SEQ ID NO: 1 under stringent conditions.

[0064] In a further aspect, the present invention relates to a BNYVV-resistant plant of the genus Beta or a part thereof, in which the nucleic acid molecule according to the invention is endogenously present, or in which one or more of the above-defined mutations have been introduced into an endogenous nucleic acid molecule otherwise correlating with the WB-sensitive genotype, wherein the plant or a part thereof does not belong to the species Beta vulgaris subsp. maritima In one embodiment, the plant according to the invention is a hybrid plant or a double haploid plant. In a further embodiment of the plant according to the invention, the nucleic acid molecule or the one or more mutations are heterozygous or homozygous as defined above. In the case of, for example, a hybrid plant, the nucleic acid molecule or the one or more mutations can also be hemizygous.

[0065] Furthermore, the application discloses that the plant additionally comprises, transgenically or endogenously, a second nucleic acid molecule at a different or further position in the genome, which encodes a polypeptide capable of conferring resistance to BNYVV in the plant in which the polypeptide is expressed as a result of transcription of the second nucleic acid molecule. A different or further position in the genome may mean that the second nucleotide molecule is located outside the chromosomal interval of chromosome III from s3e4516s05 to s3e5918s01. For example, if not already present in the starting genotype, the RZ-3 gene described in international application WO 2014 / 202044 A1 may be introduced into the wb -R plant of the present plant. In the present application, therefore, wb -R / RZ-3-R plants which are preferably homozygous for one and particularly preferably for both resistance genes. In this context, it is understood that due to the provision of sequence information for the wb -R gene in the present application and, for example, the sequence information for the RZ-3 gene described in the aforementioned WO 2014 / 202044 A1, such doubly resistant plants can be obtained for the first time solely through crossing and marker-assisted selection, so that no genetic engineering techniques are required.

[0066] Accordingly, the present invention also relates to a method for identifying a plant of the genus Beta which is resistant to the pathogen BNYVV, characterized in that the method comprises the following step: (i) detecting the presence of said nucleic acid molecule, wherein the polypeptide encoded thereby has (i) a glutamine (Q) at the position corresponding to position 307 in the reference amino acid sequence SEQ ID NO: 2, or (ii) an arginine (R) at the position corresponding to position 437 in the reference amino acid sequence SEQ ID NO: 2, and / or the expression of said nucleic acid molecule, wherein the nucleic acid molecule has the following nucleotide substitutions at the positions corresponding to the positions in the reference nucleotide sequence SEQ ID NO: 1: (a) C instead of A at position 919, or (b) G instead of A at position 1310; in the plant ora sample thereof; and / or (ii) detecting the presence of said nucleic acid molecule, wherein the polypeptide encoded thereby has (i) a glutamine (Q) at the position corresponding to position 307 in the reference amino acid sequence SEQ ID NO: 2, and (ii) an arginine (R) at the position corresponding to position 437 in the reference amino acid sequence SEQ ID NO: 2, and / or expressing said nucleic acid molecule, wherein the nucleic acid molecule has the following nucleotide substitutions at the positions corresponding to the positions in the reference nucleotide sequence SEQ ID NO: 1: (a) C instead of A at position 919, and (b) G instead of A at position 1310; in the plant or a sample thereof, and optionally (iii) selecting the BNYVV-resistant plant.

[0067] As disclosed in the present application, the method may advantageously comprise the detection of at least one polymorphism resulting in an amino acid exchange compared to the Figur 1B (SEQ ID NO: 2) shown amino acid sequence, preferably at one of the Abbildung 1D (SEQ ID NO: 4) shown amino acid sequence highlighted (see also the figure legend to Abbildung 1 ) with the Figur 1C (SEQ ID NO: 3) using molecular markers that detect the polymorphisms, in particular diagnostic polymorphisms. This detection preferably takes place using at least one molecular marker per polymorphism, in particular per diagnostic polymorphism. The person skilled in the art knows which marker techniques are to be used to detect a corresponding polymorphism and how to construct molecular markers therefor. Furthermore, the present application discloses molecular markers that detect a polymorphism according to Figur 1C oder D describe or detect, as well as the use of a molecular marker to detect a polymorphism according to Figur 1C and / or D.

[0068] Furthermore, the above identification methods also represent methods for selecting a plant that exhibits resistance to BNYVV. The selection method comprises a final step of selecting a resistant plant.

[0069] Furthermore, upstream of the wb -R gene (as SEQ ID NO: 3) adjacent genomic DNA sequence segments and downstream to the wb -R gene adjacent genomic DNA sequence sections, which are located in the immediate vicinity, preferably on chromosome III and therefore closely linked to the wb -R gene are used as DNA regions for the development of diagnostic markers for wb -R can be used.

[0070] Therefore, the present invention relates to a method for selecting a plant which has resistance to BNYVV.

[0071] As disclosed in the present application, the method for selection may comprise the use of a molecular marker on a DNA sequence according to SEQ ID NO: 3 and / or on a DNA sequence located in the immediate vicinity, preferably on chromosome III. Preferably, the markers located in the immediate vicinity, according to the markers s3e4516s05 and s3e5918s01 described in the examples, are within a range of 0.11 cM, which corresponds to a physical length of approximately 100,000 bp, of the wb-R gene, and exhibit a comparable diagnostic value (DW) to (a) s3e4516s05_cyt / DW=0.89; left flanking and (b) s3e5918s01_ade / DW=0.91; right flanking. The method described in the present application further typically comprises a final step of selecting a resistant plant. The person skilled in the art knows how to develop and use markers based on the disclosed sequence information.

[0072] Accordingly, the present invention also relates to a plant, advantageously in particular a BNYVV-resistant plant or a part thereof, which has been identified and optionally selected by a method as described above.

[0073] In particular, the present invention relates to a population of plants comprising plants which are obtainable according to one of the methods according to the invention described above and are preferably resistant to root beard disease (rhizomania) or BNYVV infection, and are characterized by the presence of a nucleic acid molecule according to the invention. The population preferably comprises at least 10, preferably 50, more preferably 100, particularly preferably 500, and in particular in agricultural cultivation preferably at least 1000 plants. Preferably, the proportion of plants in the population which do not carry the nucleic acid molecule according to the invention and / or are susceptible to root beard disease is less than 25%, preferably less than 20%, more preferably less than 15%, even more preferably 10%, and particularly preferably less than 5%, 4%, 3%, 2%, 1%, or 0.5%, if present at all.

[0074] As disclosed in the present application, the following advantages can also be achieved for the breeding and development of new resistant plant lines of the genus Beta: sequence information as well as the identified polymorphisms, which allow discrimination between resistant wb -R- and susceptible wb The s-alleles of the revealed gene allow for marker development directly within the gene, which represents a significant benefit for plant breeders, particularly with regard to the development of optimized elite lines without linkage drag. Furthermore, knowledge of the sequential structure can be used to identify other new resistance genes, particularly those against rhizomania, which may be partially homologous or orthologous, for example.

[0075] The use of the resistant gene allele disclosed here in cis- or transgenic approaches opens up the possibility of developing new resistant varieties of the Beta genus that exhibit higher resistance based on the dose effect or in which stacking the disclosed gene with other resistance genes, particularly with the RZ-3 gene, can prevent resistance breakage and optimize resistance expression. Furthermore, modifications of the gene using TILLING or targeted genome engineering to develop new resistance alleles are possible.

[0076] As disclosed in the present application, the identified resistant wb-R gene allele in a genetic or molecular stack with other genetic elements that can confer agronomically advantageous traits in a plant. This can significantly increase the economic value of crops, for example by increasing yield or opening up new areas for a plant that were previously inaccessible to the cultivation of this plant due, among other things, to biotic factors such as strong pathogen pressure or abiotic factors such as drought. In particular, the present invention relates to the use of the identified resistant wb-R gene allele in methods for controlling infestation with the pathogen Beet Necrotic Yellow Vein Virus (BNYVV) in the agrocultural or horticultural cultivation of plants of the genus Beta, for example comprising identifying and selecting plants of the genus Beta by means of one of the methods described above, cultivating the plants thus selected or progeny thereof.

[0077] An agronomically advantageous trait, for example, is tolerance to a herbicide such as glyphosate, glufosinate, or ALS inhibitors. Numerous other herbicides and their applicability are known to the skilled person from the prior art. They can draw on the prior art to gain knowledge of which genetic elements are to be used in which way to implement a corresponding tolerance in plants. Another example of an agronomically advantageous trait is additional pathogen resistance, where pathogens can be, for example, insects, viruses, nematodes, bacteria, or fungi. By combining different pathogen resistances / tolerances, a broad pathogen defense for a plant can be achieved, since genetic elements can have complementary effects. For this purpose, the skilled person is familiar with numerous resistance genes, for example, as genetic elements.Another example of an agronomically advantageous trait is chilling or frost tolerance. Plants exhibiting this trait could be sown earlier in the year or could remain in the field longer, for example, during frost periods, which could lead to increased yields. Here, too, the skilled person can draw on the state of the art to find suitable genetic elements. Further examples of agronomically advantageous traits are water use efficiency, nitrogen use efficiency, and yield. Genetic elements that can be used to confer such traits could be found in the state of the art.

[0078] Furthermore, numerous modifications for pathogen defense are known to the person skilled in the art. In addition to the frequently described families of R genes, the Avr / R approach, Avr gene complementation (WO 2013 / 127379), the autoactivation of an R gene (WO 2006 / 128444), the HIGS (host induced gene silencing) approach (e.g. WO2013 / 050024) or the VIGS (virus induced gene silencing) approach could be advantageously used. In particular, the autoactivation of an R gene could be of importance for the present invention. For this purpose, a nucleic acid is to be created that encodes an autoactivated resistance protein for generating resistance to pathogens in plants. This nucleic acid then has only a limited part of an NBS-LRR resistance gene such as the wb-R gene extending from the 5' end of the coding region of the NBS-LRR resistance gene downstream to the beginning of the NBS domain of the NBS-LRR resistance gene, wherein the NBS-LRR resistance gene is not a TIR-NBS-LRR resistance gene.

[0079] Furthermore, the present application also discloses the use of the resistant wb -R gene alleles , identified by a method described above, for combination with a modification described above, or with a genetic element described above, which can confer one or more agronomically advantageous traits in a plant.

[0080] Furthermore, in addition to the plant according to the invention, the present application also discloses seeds or offspring, or organs, plant parts, tissues, or cells thereof in the production of products typically made from renewable raw materials, such as food and animal feed, preferably sugar or syrup (molasses). The molasses is also used for industrial applications, for example in alcohol production or as a nutrient medium for the production of biotechnological products, in the production of materials or substances for the chemical industry, e.g., fine chemicals, pharmaceuticals or precursors thereof, diagnostics, cosmetics, bioethanol, or biogas. An example of the use of sugar beet as a biogenic raw material in biogas plants is described in the application DE 10 2012 022 178 A1, see, for example, paragraph 10.

[0081] The following examples illustrate the invention. Unless otherwise stated, standard molecular biological methods were used, see, for example, (Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001), Fritsch et al. , Cold Spring Harbor Laboratory Press: 1989; Mayer et al., Immunochemical Methods In Cell And Molecular Biology, eds., Academic Press, London, 1987) and Weir et al., Handbook Of Experimental Immunology, Volumes I-IV, Blackwell, eds., 1986). EXAMPLES Example 1: Identification of a gene conferring resistance to rhizomania (root beard, WB) (WB1) and associated sensitive alleles

[0082] In a population of sugar beet (Beta vulgaris L.) with an introgression from an accession of Beta vulgaris subsp. maritimaIn this introgression, a gene or genome segment conferring resistance to rhizomania was detected using markers with good diagnostic value (DW): (a) s3e4516s05_cyt / DW=0.89; left flanking and (b) s3e5918s01_ade / DW=0.91; right flanking. However, these two markers are not fully diagnostic due to the occurrence of null alleles in different pedigrees. The genomic region between s3e4516s05 and s3e5918s01 encompasses a genetic length of 0.11 cM, which corresponds to a physical length of approximately 100,000 bp. This sequence region is highly repetitive and shows strong structural variation in different genotypes; therefore, it is very difficult to develop further diagnostic markers. Furthermore, due to the lack of knowledge of the genetic structure of the genome segment conferring rhizomania resistance, it is only possible to a limited extent to further reduce the potential "negative linkage drag" around the causal gene.

[0083] Initial experiments to narrow down the relevant genome segment and possibly locate a gene mediating the observed resistance or a gene locus responsible for the rhizomania-sensitive trait of the plants failed, partly because the target region is highly repetitive and shows strong structural variation (null alleles) in many genotypes, and other markers with high diagnostic value were not available. Furthermore, expression analysis of candidate genes in the target region initially provided no evidence, i.e., a specific response to rhizomania infection.Finally, the phenotyping of the plants proved difficult because the resistance expression was not always clear for unknown reasons, so that initially the presence of multigenic resistance and / or epigenetic effects was suspected, which could only be ruled out by increasing the number of plants examined with 90-180 offspring and with intensive statistical procedures (t-test, power analysis).

[0084] In further experiments and analyses, using "map-based cloning", which included the steps of genetic fine mapping, physical mapping, WHG (whole genome) sequence analysis, establishment of a very large segregating population of over 2000 F2 offspring, recombinant screening, marker development in the target region, comparative BAC sequencing in resistant (RR) and sensitive / susceptible (ss) genotypes, bioinformatics analyses, protein predictions, and comparison of the proteins, it was determined that an NB-ARC (NBS-LRR) gene is responsible for the observed rhizomania resistance.This NBS-LRR gene was identified through intensive fine mapping. Due to sequence complexity, the assembly of the RR and ss sequences was not readily apparent. The NBS-LRR gene could only be precisely identified by analyzing the progeny of the four closest recombinant plants (two direct recombinants on the left and two direct recombinants on the right surrounding the gene). The sequence of the NBS-LRR gene of the resistant genotype is shown in SEQ ID NO: 3, and the corresponding gene or genotype is called "wb-R" for "root beard resistance." A total of 17 "non-synonymous" single nucleotide polymorphisms (SNPs) (polymorphisms that result in an amino acid substitution in the protein) were found in the NBS-LRR gene. Based on sequence data consisting of sensitive and resistant genotypes, 5 amino acid substitutions were shown to be fully diagnostic: . K307Q, "C" instead of "A" in the genomic sequence at position 919 in the resistant genotype, which replaces the encoded lysine (K) at position 307 of the susceptible gene with a glutamine (Q), Q437R, "G" instead of "A" in the genomic sequence at position 1310 in the resistant genotype, which replaces the encoded glutamine (Q) at position 437 of the susceptible gene with an arginine (R), R566H, "A" instead of "G" in the genomic sequence at position 1697 in the resistant genotype, which replaces the encoded arginine (R) at position 566 of the susceptible gene with a histidine (H), Q731K, "A" instead of "C" in the genomic sequence at position 2191 in the resistant genotype, which replaces the encoded glutamine (Q) at position 731 of the susceptible gene with a lysine (K), and P831S, "T" instead of "C" in the genomic sequence at position 2491 in the resistant genotype, which replaces the encoded proline (P) at position 831 of the susceptible gene with a serine (S); see Figure 1Based on the recombination points, one or both of the amino acid substitutions K307Q or Q437R can be considered causal for conferring resistance. The corresponding gene sequences or genotypes that correlate with the root beard-sensitive phenotype - also "wb -s " for "root beard-sensitive". Example 2: Validation of the wb-R gene using RNAi approach

[0085] In addition to the verification of the gene using close recombinants described above, the resistance effect of the gene can be demonstrated using RNA interference as a further means of proof; see, for example, the verification of the RZ-3 gene described in the examples of international application WO 2014 / 202044 A1 or the verification of the vil gene described in the examples of international application WO 2011 / 032537 A1. For this purpose, a resistant standard sugar beet genotype is transformed with a DNA construct encoding a double-stranded hairpin RNA. This dsRNA is capable of post-transcriptional gene silencing, which would reduce or eliminate the effect of the resistant wb-R gene allele, thus rendering the previously resistant sugar beet genotype sensitive to rhizomania.

[0086] To prepare a suitable DNA construct, a defined target sequence region of the resistant wb-R gene allele, for example, 400-500 base pairs in length, preferably selected from a region of the coding sequence specific for the resistant wb-R gene allele, is amplified by PCR and cloned in both the sense and antisense directions into the vector pZFN, which is suitable for the synthesis of hairpin structures (see Fig. 6 of the international application WO 2014 / 202044 A1). This vector has a double CaMV 35S promoter, a multiple cloning site, an intron from the gene AtAAP6, which is encoded in Arabidopsis thalianaencodes an amino acid permease, has another multiple cloning site and a nos terminator. The transformation of the sugar beets with the provided vector is carried out according to the protocol of Lindsey & Gallois, J. Exp. Bot. 41 (1990), 529-536, using the antibiotic kanamycin as a selection marker. After several selection steps, the successful transformation of transgenic shoots is confirmed by PCR by detecting the presence of the nptII gene, the AAP6 intron and the two t-DNA border sequences (LB / RB) and the absence of vir Positive rungs are in vitroThe plants are clonally propagated to 30 shoots each, rooted, and transferred to soil in the greenhouse. Approximately two weeks later, the transgenic sugar beet plants are pricked out into rhizomania-contaminated soil, where they are cultivated for eight to ten weeks. Non-transformed plants from the same resistant genetic standard transformation background are used as controls under the same conditions. To detect the severity of rhizomania, the roots of the sugar beet plants are harvested, and BNYVV infection is quantified using an ELISA test, with a low ELISA value indicating resistance and a high value indicating sensitivity (Mechelke 1997, supra; Clark & Adams, J. Gen. Virol. 34 (1977), 475-483). As expected, the ELISA value of the transformed sugar beet is significantly higher with a mean value of 3-4 than the ELISA value of the still resistant control with a mean value of 1-2 and comparable to the sensitive standard.The results of the ELISA test can then show that the specific gene silencing of the resistant . wb -R allele in the transformation background, a previously resistant plant becomes sensitive to BNYVV. Consequently, the wb -R gene of the present invention can be clearly verified as the resistance gene.

[0087] The validation of the gene function can also be achieved by complementing a WB sensitive plant, in particular sugar beet, with a wb-R gene, for example, by transforming a plant expression vector containing a nucleic acid molecule with the nucleotide sequence of SEQ ID NO: 3 or an equivalent nucleotide sequence encoding a polypeptide with an amino acid sequence of SEQ ID NO: 4, each under the control of a constitutive promoter, into a sensitive genotype. For the transformation, the pZFN vector and the techniques mentioned can generally be used, or also those described above in the general description.

Claims

1. Nucleic acid molecule encoding a polypeptide capable of mediating a resistance to a pathogen in a plant in which the polypeptide is expressed, characterized in that the nucleic acid molecule comprises a nucleotide sequence selected from (a) a nucleotide sequence encoding a polypeptide with an amino acid sequence that is at least 80% identical to the SEQ ID NO: 2, wherein the nucleic acid molecule comprises one or more mutations that cause that the polypeptide, encoded by the nucleic acid molecule, has an amino acid different from lysine (K) at the position corresponding to position 307 in the reference amino acid sequence SEQ ID NO: 2, and / or at the position corresponding to position 437 in the reference amino acid sequence SEQ ID NO: 2, comprises an amino acid distinct from glutamine (Q); and (b) a nucleotide sequence that hybridizes with the complementary sequence to SEQ ID NO: 1 under stringent conditions, wherein the nucleotide sequence has one or more of the following nucleotide substitutions at those sites corresponding to the sites in the reference nucleotide sequence SEQ ID NO: 1: C instead of A at position 919; G instead of A at position 1310; A instead of G at position 1697; A instead of C at position 2191; and / or T instead of C at position 2491; preferably wherein the pathogen is the beet necrotic yellow vein virus (BNYVV) and the plant is a plant of the genus Beta, preferably sugar beet (Beta vulgaris L.).

2. The nucleic acid molecule according to claim 1, characterized in that the encoded polypeptide further (i) has an amino acid different from arginine (R) at the position corresponding to position 566 in the reference amino acid sequence SEQ ID NO: 2, and / or (ii) at the position corresponding to position 731 in the reference amino acid sequence SEQ ID NO: 2, which is distinct from glutamine (Q), and / or (iii) at the position corresponding to position 831 in the reference amino acid sequence SEQ ID NO: 2, has an amino acid distinct from proline (P).

3. The nucleic acid molecule according to claim 1 or 2, characterized in that the encoded polypeptide (i) has a glutamine (Q) at the position corresponding to position 307 in the reference amino acid sequence SEQ ID NO: 2, and / or (ii) has an arginine (R) at the position corresponding to position 437 in the reference amino acid sequence SEQ ID NO: 2, and / or (iii) has a histidine (H) at that position, which corresponds to position 566 in the reference amino acid sequence SEQ ID NO: 2, and / or (iv) has a lysine (K) at the position corresponding to position 731 in the reference amino acid sequence SEQ ID NO: 2, and / or (v) has a serine (S) at that position corresponding to position 831 in the reference amino acid sequence SEQ ID NO: 2.

4. The nucleic acid molecule according to claim 3, characterized in that the nucleic acid molecule has one or more of the following nucleotide substitutions at those sites corresponding to the sites in the reference nucleotide sequence SEQ ID NO: 1: (a) C instead of A at position 919; (b) G instead of A at position 1310; (c) A instead of G at position 1697; (d) A instead of C at position 2191; and / or (e) T instead of C at position 2491.

5. The nucleic acid molecule according to any one of claims 1 to 4, characterized in that the nucleic acid molecule encodes a polypeptide with an amino acid sequence according to SEQ ID NO: 4 and / or comprises the coding DNA sequence according to SEQ ID NO: 3.

6. A vector comprising the nucleic acid molecule according to any one of claims 1 to 5.

7. A transgenic plant cell, preferably from a plant of the genus Beta, comprising the nucleic acid molecule according to any one of claims 1 to 5 as a transgene, optionally under the control of a heterologous promoter, or the vector according to claim 6.

8. A transgenic plant, preferably of the genus Beta, or a part thereof, comprising a plant cell according to claim 7.

9. A BNYVV-resistant plant of the species Beta vulgaris or a part thereof in which the nucleic acid molecule is endogenously present according to any one of claims 1 to 5, or in which one or more mutations, defined in any one of claims 1 to 4, into an endogenous nucleic acid molecule having a nucleotide sequence comprising the sequence according to SEQ ID NO: 1, or which is associated with the complementary sequence to SEQ ID NO: 1 which have been hybridised under stringent conditions, whereby the plant or part of it does not belong to the subspecies Beta vulgaris subsp. maritima.

10. The plant according to claim 8 or 9, characterized in that the plant is a hybrid plant or a double haploid plant and / or that the nucleic acid molecule or one or more mutations are heterozygous or homozygous.

11. A seed from the plant according to any one of claims 8 to 10, wherein the seed comprises one or more of the aforementioned nucleic acid molecules or vectors transgenically or endogenously.

12. Method for producing a BNYVV-resistant plant, comprising the following steps: (a) Mutagenization of plant cells and subsequent regeneration of plants from the mutagenized plant cells or mutagenization of plants, and (b) Identification of a plant from (a) having one or more mutations in an endogenous nucleic acid molecule in any one of the claims 1 to 5.

13. Method for identifying a plant of the genus Beta which is resistant to the pathogen BNYVV, characterized in that the method comprises the following step: (i) Evidence of the presence of a nucleic acid molecule according to claim 3, wherein the polypeptide encoded thereof (i) has a glutamine (Q) at the position corresponding to position 307 in the reference amino acid sequence SEQ ID NO: 2, or (ii) has an arginine (R) at the position corresponding to position 437 in the reference amino acid sequence SEQ ID NO: 2, and / or the expression of the nucleic acid molecule according to claim 4, wherein the nucleic acid molecule has the following nucleotide substitutions at those sites corresponding to the sites in the reference nucleotide sequence SEQ ID NO: 1: (a) C instead of A at position 919, or (b) G instead of A at position 1310; in the plant or a sample thereof; and / or (ii) Evidence of the presence of a nucleic acid molecule according to claim 3, wherein the polypeptide encoded therein (i) has a glutamine (Q) at the position corresponding to position 307 in the reference amino acid sequence SEQ ID NO: 2, and (ii) has an arginine (R) at the position corresponding to position 437 in the reference amino acid sequence SEQ ID NO: 2, and / or the expression of the nucleic acid molecule according to claim 4, wherein the nucleic acid molecule has the following nucleotide substitutions at those sites corresponding to the sites in the reference nucleotide sequence SEQ ID NO: 1: (a) C instead of A at position 919, and (b) G instead of A at position 1310; in the plant or a sample of it, and, if necessary, (iii) Selection of the BNYVV resistant plant.

14. A population of plants comprising plants according to any one of claims 9 or 10 or plants identified and, if necessary, selected by a method according to claim 13.

15. Methods for the control of infestation with the pathogen Beet Necrotic Yellow Vein Virus (BNYVV) in the agricultural or horticultural cultivation of plants of the genus Beta, comprising (I) the identification and selection of plants of the genus Beta by means of a method according to claim 13, and II) Cultivation of the plants from I) or descendants thereof.