Resistance gene against

Abstract

The present invention provides a novel nucleic acid molecule encoding a polypeptide that can confer resistance to pathogens, especially the "Beet Necrosis Yellow Vein Virus" (BNYVV), in plants, especially in plants of the genus Beta expressing the polypeptide, and also provides preferred nucleic acid molecules encoding the RZ-3 gene of Beta maritima, as well as derivatives and homologues thereof. Further aspects of the invention include vectors, transgenic plant cells, transgenic plants, transgenic plants and methods of making them, and methods of identifying nucleic acid molecules that confer resistance.

Description

[0001] This application is a divisional application of the invention patent application filed on June 6, 2014, with application number 201480045665.2 and invention title "Resistance Gene against Root Fever". Technical Field

[0002] This invention relates to nucleic acid molecules encoding polypeptides that can confer resistance to pathogens, particularly Beetula species expressing the polypeptide, in plants, especially to BNYVV. The invention also relates to polypeptides that can confer resistance to pathogens, particularly to BNYVV, in Beetula species expressing the polypeptide, and which are encoded by nucleic acid molecules of the present invention. The invention further relates to transgenic plants, plant cells, plant organs, plant tissues, plant parts, or plant seeds comprising the nucleic acid molecule or a portion thereof, and to methods for producing such transgenic plants or plant cells. The invention also includes methods for detecting the resistance-conferring nucleic acid molecule and methods for screening plants or plant cells having the resistance-conferring nucleic acid molecule. Background Technology

[0003] In terms of profitability, rhizomania is the most serious beet disease worldwide, potentially causing 50% or more of revenue loss. Also known as "root madness," it is caused by Beet Necrotic Yellow Vein Virus (BNYVV) and induced by the soil-borne protozoan *Polymyces beetula*. Polymyxa beetBNYVV infection manifests as increased proliferation in fine and secondary roots, and the formation of a significantly reduced root body with decreased sugar content. Infected plants exhibit reduced water uptake and are therefore more susceptible to drought stress. When the infection spreads throughout the plant, it causes yellowing of leaf veins, necrotic lesions, and yellow spots on the leaves. As with other viral diseases, curative treatment is not possible; the only way to prevent damage is through breeding resistant varieties. The three main genes currently identified against BNYVV are RZ-1 (also known as “Holly”), RZ-2, and RZ-3. In addition, more BNYVV resistance genes have been described in the literature, though these are of less importance. Currently, the resistance gene RZ-1 has been incorporated into most breeding lines (seed and / or pollinating parent components). However, it has been found that resistance conferred by RZ-1 is insufficient in severely infected areas or areas with multiple BNYVV pathogenicities (e.g., Sohi & Maleki, 2004). Therefore, it has been proposed in the past to use the RZ-1 in conjunction with, for example, the RZ-2 or RZ-3. The RZ-2 and RZ-3 originated from sugar beets (…). Beetroot subsp. maritime RZ-1 originates from (WB42, WB41) and is genetically located in the same region on chromosome 3 of the beet genome, while RZ-2 and RZ-3 are also located on chromosome 3, but to the south of RZ-2 and RZ-3. Scholten et al. (1999) determined a distance of 20-25 cM between the major RZ genes RZ-1 and RZ-2. Gidner et al. (2005) found a shorter distance of 5 cM between RZ-1 and RZ-2 and did not conclude that RZ-2 and RZ-3 are located at the same locus. Schmidlin et al. (2008) identified differentially induced genes through expression analysis in infected beets; however, these genes did not correspond to RZ-2 or RZ-3. In the study by Larson et al. (2008), some BNYVV-induced proteins were detected in beets using MALDI-TOF-MS; however, scientists were unable to identify proteins encoded by RZ-1, RZ-2, or RZ-3. Furthermore, the sequence region, especially the region near the resistance gene, is repetitive, making the development of diagnostic markers particularly difficult. Until now, high-resolution marker maps and confirmed candidate genes for specific root rot resistance genes have not been publicly available. Additionally, the functional background, i.e., the genetic structure, of these resistance genes was not previously fully understood.

[0004] For sustainable cultivation of BNYVV resistant plants aimed at offsetting the risk of BNYVV isolates that break resistance, it is necessary to continuously identify new resistance genes and integrate these genes into the gene pool of crop plants such as sugar beets. Summary of the Invention

[0005] This invention is developed based on the aforementioned prior art, and one object of the invention is to provide nucleic acid molecules and / or polypeptides that can confer resistance to root rot in plants. A further object is to provide transgenic root rot-resistant plants, and methods for producing them. A further object of the invention is to provide methods for using and developing molecular markers that enable the effective cultivation of root rot-resistant plants and the development of new resistant plant strains.

[0006] The embodiment of the present invention that achieves the stated objective is based on the study of a donor beet subspecies. maritime ( Beta common subsp. maritime The genetic fine localization, identification, isolation, and characterization of a gene encoding a polypeptide or protein that can confer resistance to pathogens in plants expressing the polypeptide.

[0007] Some of the terms used in this application will be explained in more detail below: When the term "approximately" is used in conjunction with a description of nucleotide sequence length, it indicates a deviation of ±200 base pairs, preferably ±100 base pairs, and especially preferably ±50 base pairs.

[0008] Plants in the genus *Betula* belong to the family Myristica fragrans (Amaranthaceae). These plants include the species *Betula macrocarpa* (large-fruited beet). Beta macrocarpa ),beet( Beetroot ), Beta lomatogona , Beetroot White beetroot ( Beta corolliflora ), Three-stalk beet ( Beta trigyna )and Dwarf beet Plants. Species Beta common plants, especially Beetroot subsp. maritime (Seemangold) or Beta common subsp. common Plants. These include, for example: Beetroot subsp. common var. al very (beets in a narrow sense) Beetroot ssp. common var. common (Mangold) Beta v common ssp. common var. condiment (beetroot) Beetroot ssp. common var. thick / white (Beet for animal feed)

[0009] The term "hybridization" or "hybridization technique" is understood as the process by which a single-stranded nucleic acid molecule attaches to a nucleic acid strand that is complementary to it to the greatest possible extent, i.e., forming base pairs. Standard methods for hybridization are described, for example, in Sambrook et al. (2001). This is preferably understood to mean that at least 60%, more preferably at least 65%, 70%, 75%, 80%, or 85%, and particularly preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the bases in the nucleic acid molecule are capable of forming base pairs with a nucleic acid strand that is complementary to it to the greatest possible extent. The likelihood of this annealing depends on the stringency of the hybridization conditions. The term "stringency" is related to hybridization conditions. High stringency is given when base pairing is difficult; low stringency is given when base pairing is easy. The stringency of hybridization conditions depends on, for example, salt concentration or ionic strength or temperature. Generally, stringency can be increased with increasing temperature and / or decreasing salt concentration. "Stringent hybridization conditions" are understood to refer to conditions under which hybridization occurs primarily between homologous nucleic acid molecules. The term "hybridization conditions" here refers not only to the general conditions during the actual attachment of nucleic acids but also to the general conditions during subsequent washing. Strict hybridization conditions are, for example, conditions primarily for hybridization only of nucleic acid molecules having at least 70%, preferably at least 75%, at least 80%, at least 85%, or at least 90%, particularly preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. Strict hybridization conditions are, for example, hybridization at 65 °C in 4 x SSCs, followed by multiple washes at 65 °C in 0.1 x SSCs for approximately 1 hour. The term "strict hybridization conditions" as used herein can also refer to hybridization at 68 °C for 16 hours in 0.25 M sodium phosphate, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA, followed by two washes at 68 °C in 2 x SSCs and 0.1% SDS. Hybridization is preferably performed under strict conditions.

[0010] "Isolated nucleic acid molecules" are understood to refer to nucleic acid molecules that have dissolved from their natural or pristine environment. This term also includes synthetically produced nucleic acid molecules. "Isolated polypeptides" are understood to refer to polypeptides that have dissolved from their natural or pristine environment. This term also includes synthetically produced polypeptides.

[0011] "Molecular markers" are nucleic acids that exhibit polymorphism in plant populations. Such markers can therefore detect and distinguish different allelic states (alleles). Known analytical methods for this purpose include RFLP, AFLP, SNP, SSR, or KASP. The term "molecular marker" refers to nucleotide sequences that are complementary to, or at least as complementary as possible to, a genomic sequence or are homologous to it, such as nucleic acids used as probes or primers. Markers describing polymorphism can be detected using established methods. These methods include, for example, PCR-based sequence-specific amplification, detection of restriction fragment length polymorphism (RFLP), detection of polynucleotide polymorphisms using allele-specific hybridization (ASH), detection of amplified variable sequences in the plant genome, detection of autonomous sequence duplication, detection of simple sequence repeats (SSR), detection of single nucleotide polymorphisms (SNP), or detection of amplified fragment length polymorphism (AFLP). Furthermore, methods for detecting expressed sequence tags (ESTs) and SSR markers derived from EST sequences, and random amplified polymorphic DNA (RAPD) are also known.

[0012] A "promoter" is a non-translated regulatory DNA sequence, usually located upstream of the coding region, which contains the binding site of RNA polymerase and initiates DNA transcription.

[0013] A "pathogen" is an organism that interacts with a plant and causes disease symptoms in one or more organs of the plant. These pathogens include, for example, animals, fungi, bacteria, viruses, or oomycetes.

[0014] "Pathogen infection" is understood as the earliest moment when a pathogen interacts with the host plant tissue. Taking the viral pathogen BNYVV as an example, this virus is caused by the protozoan beet polymyxa (…). Polymyxa beta e (Spread by) Polymyxin B ( Polymyx The virus forms spores that can survive underground for decades. These spores also survive. When these dormant spores germinate to form mobile zoospores, the virus can penetrate the cells of the plant host tissue through these spores and interact with the host there (Esser 2000).

[0015] Plant "organs" refer to, for example, leaves, branch axes, stems, roots, hypocotyls, vegetative buds, meristems, embryos, anthers, ovules, or fruits. Plant "parts" refer to a combination of plant organs, such as flowers and seeds, or a portion of an organ, such as a cross section of a stem. Plant "tissues" are, for example, callus tissue, storage tissue, meristems, leaf tissue, stem tissue, root tissue, plant tumor tissue, or reproductive tissue. Plant "cells" are, for example, understood to mean isolated cells with cell walls or aggregates of them, or protoplasts.

[0016] The term "resistance" should be understood broadly and encompass protection ranging from delaying disease progression to completely inhibiting it. An important example of a pathogen is beet necrosis virus (BNYVV). The resistant plant cells or resistant plants of the present invention preferably achieve resistance to BNYVV. Resistance to a pathogen should be equivalent to resistance to the disease caused by that pathogen, such as resistance to BNYVV and resistance to rhizomatous root rot.

[0017] In this document, "transgenic plant" refers to a plant whose genome has integrated at least one nucleic acid. The at least one nucleic acid described herein can be a heterologous nucleic acid. Preferably, the nucleic acid is integrated in a stable manner, meaning that the integrated nucleic acid is stably maintained in the plant, can be expressed, and can be stably passed on to offspring.

[0018] This invention discloses a nucleic acid molecule comprising a polypeptide, said polypeptide being capable of conferring resistance to pathogens in plants expressing the polypeptide. The nucleic acid molecule comprises a nucleotide sequence selected from the following: a) A nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3; b) The nucleotide sequence containing the coding sequence of the DNA sequence of SEQ ID NO: 1; c) A nucleotide sequence that hybridizes with the complementary sequence of the nucleotide sequence of a) or b) under stringent conditions; d) A nucleotide sequence encoding a polypeptide, wherein the polypeptide is derived from the polypeptide encoded by the nucleotide sequence of a) or b) by substitution, deletion and / or addition of one or more amino acids in the amino acid sequence encoded by the nucleotide sequence of a). e) A nucleotide sequence encoding a polypeptide having at least 60% identical amino acid sequence to the amino acid sequence encoded by the nucleotide sequence of a) or b); f) A nucleotide sequence encoding at least one nucleotide-binding domain (NBS) corresponding to amino acid positions 168-227 of SEQ ID NO: 2 or amino acid positions 182-241 of SEQ ID NO: 3, at least one leucine-rich domain (LRR) corresponding to amino acid positions 591-613 of SEQ ID NO: 2 or amino acid positions 605-627 of SEQ ID NO: 3, and / or at least one internal repeating domain (IR) corresponding to amino acid positions 1013-1072 of SEQ ID NO: 2 or amino acid positions 1027-1086 of SEQ ID NO: 3.

[0019] The nucleic acid molecule may be an isolated nucleic acid molecule. It is preferably DNA, and particularly preferably cDNA (encoding DNA). The polypeptide encoded by the nucleic acid molecule of the present invention preferably confers resistance to the viral pathogen “beet necrosis yellow vein virus” (BNYVV), which causes root rot in plants. Furthermore, the polypeptide encoded by the nucleic acid molecule of the present invention, especially in beet plants, confers resistance to the pathogen. The plant is preferably beet (…). Beetroot Plant species, especially subspecies. Beetroot subsp. maritime or Beetroot subsp. common Plants; these include, for example, crop types consisting of sugar beets, beetroot, fodder beets, leaf beets, Swiss chard, etc.

[0020] In one embodiment of the nucleic acid molecule of the present invention, the nucleic acid molecule comprises the nucleotide sequence of a). The amino acid sequence of SEQ ID NO: 2 of the encoded polypeptide and / or the amino acid sequence of SEQ ID NO: 3 of the encoded polypeptide constitutes the resistance protein of the RZ-3 gene. In this document, this is an NBS-LRR type resistance gene protein characterized by a specific structural motif. The general structure of such resistance proteins in plants has been well studied (Martin). et al. (2003). However, the mechanism of structure formation, particularly the so-called LRR domain (a potential recognition domain for most unknown pathogenic agents), remains unpredictable. Therefore, it is impossible to identify BNYVV resistance-contributing genes or proteins purely based on known structural motifs. Identification of RZ-3 resistance genes through map-based cloning requires intensive genetic mapping and fine localization of the initially suspected target regions of the RZ-3 resistance gene. The development work will be described in more detail below.

[0021] The identified resistance protein belongs to the NBS-LRR type and has a nucleotide-binding domain (NBS, also known as NB-ARC) corresponding to amino acid positions 168-227 of SEQ ID NO: 2 or amino acid positions 182-241 of SEQ ID NO: 3 (a nucleotide-binding linker shared by APAF-1, R protein, and CED-4), a leucine-rich domain (LRR) corresponding to amino acid positions 591-613 of SEQ ID NO: 2 or amino acid positions 605-627 of SEQ ID NO: 3, and / or at least one internal repeating domain (IR) corresponding to amino acid positions 1013-1072 of SEQ ID NO: 2 or amino acid positions 1027-1086 of SEQ ID NO: 3. The NBS domain is encoded by nucleotides 2019-2882 of SEQ ID NO: 1, the LRR domain is encoded by nucleotides 3288-3356 of SEQ ID NO: 1, and the IR domain is encoded by nucleotides 4554-4871 of SEQ ID NO: 1. The NB-ARC domain is a central nucleotide-binding domain. It is likely a functional ATPase domain, expected to regulate the activity of resistance proteins. The NB-ARC domain consists of three subdomains: NB, ARC1, and ARC2. Characteristic motifs of the NB-ARC domain are APAF-1 (apoptotic protease-activator-1), hhGRExE, Walker-A or P-loop, Walker-B, GxP, RNBS-A to D, and MHD (Ooijen et al., 2008), which are believed to be responsible for hypersensitivity responses. Some of these motifs have been identified. In another embodiment of the nucleic acid molecule of the present invention, the nucleic acid molecule comprises the nucleotide sequence described in b). The nucleotide sequence comprises the coding sequence of the DNA sequence of SEQ ID NO: 1, which encodes the amino acid sequences of SEQ ID NO: 2 and 3.

[0022] In another embodiment of the nucleic acid molecule, the nucleic acid molecule comprises a nucleotide sequence of d). This nucleotide sequence encodes a polypeptide, which is a derivative of the polypeptide encoded by the nucleotide sequence of a) or b). The polypeptide derivative comprises a derived amino acid sequence having at least one or more amino acid substitutions, deletions, or additions, wherein the functionality of the encoded polypeptide / protein is preserved. If an amino acid is substituted for another amino acid having the same or similar physicochemical properties, it is referred to as a "conservative exchange" or a "semi-conservative exchange." The physicochemical properties of the amino acid are, for example, hydrophobicity or charge. Those skilled in the art know which amino acid substitutions constitute a conserved or semi-conservative exchange. Common knowledge in the art further enables those skilled in the art to identify, characterize, and detect amino acid deletions and additions that are detrimental to the functionality of the resistance protein RZ-3, and their possible locations. Those skilled in the art know that in modifications (substitution, deletion, or addition of one or more amino acids) to the amino acid sequence of the NBS-LRR protein of the present invention, the functionality of the conserved domains defined above must be preserved in particular, and therefore only a limited number of modifications of the above types are possible in these domains. When the nucleotide sequence is homologous to or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence described in a) or b), the nucleotide sequence of this embodiment encodes a derivative or derived amino acid sequence. Such a nucleotide sequence encoding a derivative or derived amino acid sequence is preferably generated directly or indirectly (e.g., by amplification or replication steps) from a starting nucleotide sequence corresponding to the full length or at least a portion of the other sequence disclosed herein, corresponding to SEQ ID NO: 1.

[0023] In another embodiment of the nucleic acid molecule of the present invention, the nucleic acid molecule comprises the nucleotide sequence of e). This nucleotide sequence encodes a polypeptide having an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, or 99% identical to the amino acid sequence encoded by the nucleotide sequence of a) or b).

[0024] In another embodiment of the nucleic acid molecule of the present invention, the nucleic acid molecule comprises the nucleotide sequence of f). The nucleotide sequence here encodes at least one nucleotide-binding domain (NBS) corresponding to amino acid positions 168-227 of SEQ ID NO: 2 or amino acid positions 182-241 of SEQ ID NO: 3, at least one leucine-rich domain (LRR) corresponding to amino acid positions 591-613 of SEQ ID NO: 2 or amino acid positions 605-627 of SEQ ID NO: 3, and / or at least one internal repeating domain (IR) corresponding to amino acid positions 1013-1072 of SEQ ID NO: 2 or amino acid positions 1027-1086 of SEQ ID NO: 3. The nucleotide sequence preferably encodes a polypeptide comprising at least one nucleotide-binding domain (NBS) corresponding to amino acid positions 168-227 of SEQ ID NO: 2 or amino acid positions 182-241 of SEQ ID NO: 3, at least one leucine-rich domain (LRR) corresponding to amino acid positions 591-613 of SEQ ID NO: 2 or amino acid positions 605-627 of SEQ ID NO: 3, and at least one internal repeating domain (IR) corresponding to amino acid positions 1013-1072 of SEQ ID NO: 2 or amino acid positions 1027-1086 of SEQ ID NO: 3. These domains are particularly preferably arranged sequentially from the N-terminus to the C-terminus in the order NBS-LRR-IR, wherein one or more amino acids may be present between each domain.

[0025] This invention also relates to polypeptides that can confer resistance to pathogens in plants expressing the polypeptide, and which are encoded by the nucleic acid molecules of this invention, wherein the pathogen is preferably BNYVV and / or the plant is preferably a beet, particularly Beetroot The polypeptide is a plant species. The polypeptide is particularly preferably having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3. The polypeptide may be an isolated polypeptide.

[0026] Another aspect of the present invention relates to vectors comprising the nucleic acid molecules of the present invention. The vector may be a plasmid, granule, bacteriophage, or expression vector, transformation vector, shuttle vector, or cloning vector; it may be double-stranded or single-stranded, linear or circular, or may be transformed into a prokaryotic or eukaryotic host in a manner that integrates into the genome or extrachromosomally. The nucleic acid molecules of the present invention are preferably operatively linked in an expression vector to one or more regulatory sequences to allow transcription and, optionally, expression in prokaryotic or eukaryotic host cells. For example, the nucleic acid molecules are regulated by a suitable promoter or terminator. Suitable promoters may be constitutively inducible promoters (e.g., the 35S promoter derived from cauliflower mosaic virus (Odell et al. 1985)), and particularly suitable are pathogen-induced promoters (e.g., the PR1 promoter derived from parsley (Rushton et al., 1996)). Particularly suitable pathogen-induced promoters are synthetic or chimeric, not naturally occurring, and composed of several elements. They contain a minimal promoter and at least one cis-regulatory element upstream of the minimal promoter, serving as a binding site for a specific transcription factor. Chimeric promoters are designed as needed 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 the nos terminator (Depicker et al., 1982).

[0027] In addition to the vectors described above, this invention also provides a method for introducing the described vectors into host cells. The vectors can be introduced by methods such as conjugation, mobilization, gene gun transformation, Agrobacterium-mediated transformation, transfection, transduction, vacuum permeation, or electroporation. Those skilled in the art are familiar with these methods and the methods for preparing the described vectors (Sambrook et al. 2001).

[0028] Another aspect of the invention relates to a host cell comprising the nucleic acid molecule or the vector of the invention. In the sense of the invention, the host cell can be a prokaryotic cell (e.g., a bacterial cell) or a eukaryotic cell (e.g., a plant cell or a yeast cell). The host cell is preferably an Agrobacterium carrying the nucleic acid molecule or vector of the invention (e.g., Agrobacterium tumefaciens). Agrobacterium tumefaciens ) or Agrobacterium tumefaciens ( Agrobacterium rhizogenesPlant cells. Those skilled in the art are familiar with many methods such as conjugation and electroporation, through which they can introduce the nucleic acid molecules or vectors of the present invention into Agrobacterium; those skilled in the art are also familiar with various transformation methods (gene gun transformation, Agrobacterium-mediated transformation), through which they can introduce the nucleic acid molecules or vectors of the present invention into plant cells (Sambrook et al. 2001).

[0029] Another aspect of the invention relates to transgenic plant cells comprising the nucleic acid molecules of the invention as transgenics or comprising vectors of the invention. Such transgenic plant cells are, for example, plant cells transformed (particularly in a stable manner) with the nucleic acid molecules of the invention or vectors of the invention. In a preferred embodiment of the transgenic plant cells, the nucleic acid molecules are operatively linked to one or more regulatory sequences to allow transcription and, optionally, expression in the plant cells. The complete construct from the nucleic acid molecules of the invention, along with the regulatory sequences, then constitutes the transgenic organism. Such regulatory sequences are, for example, promoters or terminators. Functional promoters and terminators available in many plants are known to those skilled in the art. Transgenic plant cells of the invention, particularly cells of *Sacchariformis*, preferably exhibit stronger resistance to pathogens (particularly BNYVV) compared to non-transgenic plant cells. The level of resistance to, for example, BNYVV can be qualitatively defined in *Sacchariformis* using a score-based rating method (a score-based rating system for *Sacchariformis* is known from the prior art, for example, for sugar beets, Mechelke (1997)). Higher resistance means that resistance is improved by at least one, two, three, or more score ratings. Furthermore, the present invention also relates to methods for generating transgenic plant cells of the present invention, including the step of introducing the nucleic acid molecules of the present invention or the vectors of the present invention into plant cells. For example, the introduction can be carried out by transformation, preferably by stable transformation. Suitable introduction techniques, such as gene gun transformation, Agrobacterium-mediated transformation, or electroporation, are well known to those skilled in the art (Sambrook et al. 2001).

[0030] On the other hand, the present invention relates to a transgenic plant or a portion thereof comprising the aforementioned transgenic plant cells. This portion may be a cell, tissue, organ, or a combination of several cells, tissues, or organs. A combination of several organs may be, for example, a flower or a seed. In one specific embodiment, the present invention relates to seeds from said transgenic plant, said seeds containing the nucleic acid molecule of the present invention as a transgene. The transgenic plants of the present invention, particularly plants of the genus *Sacchariformis*, preferably exhibit higher resistance to pathogens (especially BNYVV) compared to non-transformed plants (plants without said transgene). The level of resistance to, for example, BNYVV can be qualitatively defined in *Sacchariformis* plants using a rating score method (a rating score system for *Sacchariformis* plants is known from the prior art, for example, sugar beet Mechelke (1997)). Higher resistance means that resistance is improved by at least one, two, three, or more rating scores. The present invention also relates to a method for producing the transgenic plant of the present invention, comprising the step of introducing the nucleic acid molecule of the present invention or the vector of the present invention into plant cells, and optionally selecting transgenic cells. Furthermore, this method for producing transgenic plants is characterized by subsequent steps including regenerating the transgenic plant from the transgenic cells produced in the first step. Methods of regeneration are known to those skilled in the art from the prior art.

[0031] On the other hand, the present invention also relates to a method for conferring or enhancing resistance to pathogens, particularly BNYVV, in plants, especially beet plants, comprising transforming plant cells with the nucleic acid molecule of the present invention or the vector of the present invention. This method results in an improvement in resistance by at least one fractional rating, preferably two, three, or more fractional ratings. Fractional rating schemes for beet plants are known techniques, for example, for sugar beets (mechelke (1997)). In another aspect, the present invention relates to a promoter regulatory sequence for controlling the expression of a gene comprising a nucleic acid molecule of the present invention, characterized in that the regulatory sequence is capable of conferring or regulating the expression of a foreign DNA sequence due to pathogen infection, and the regulatory sequence comprises a nucleic acid molecule having a nucleotide sequence having SEQ ID NO: 1 nucleotides 1-1403. The foreign DNA sequence is preferably a nucleotide sequence encoding a plant pathogen defense component (e.g., a resistance gene (R-gene) or a gene encoding an enzyme involved in signal transduction such as a kinase or phosphatase and a G-protein), or encoding a pathogenic effector (known as an avirulence gene (avr)). Furthermore, the present invention includes recombinant DNA comprising the above-described regulatory sequence. The recombinant DNA molecule is preferably operatively linked to a heterologous DNA sequence.

[0032] Another aspect of the present invention relates to host cells transformed with the regulatory sequences described above or with the specific recombinant DNA molecules described therein, and to transgenic plants, plant tissues, or plant cells containing the regulatory sequences or recombinant DNA molecules as transgenes. The present invention also provides a method for producing transgenic plant cells, comprising the steps of introducing the regulatory sequences or recombinant DNA molecules of the present invention, and optionally selecting transgenic plant cells. The present invention further provides a method for producing transgenic plants, comprising the steps of introducing the regulatory sequences or recombinant DNA molecules of the present invention into plant cells, and optionally selecting transgenic plant cells. Such methods for producing transgenic plants are further characterized by a subsequent step of regenerating transgenic plants from the transgenic plant cells produced in the first step.

[0033] As mentioned above, the RZ-3 resistance gene was identified using map-based cloning. The methods employed include, for example, the following steps: fine genetic mapping, physical mapping, constructing a very large splice colony of over 8000 F2 splicing progeny, recombinant screening, marker development in the target region, comparative BAC sequencing in resistant and susceptible genotypes, bioinformatics analysis, protein prediction, and protein comparison. Such arduous work is very costly, and it is uncertain whether it has truly succeeded in identifying the gene. (The remaining text appears to be incomplete and requires further context.) Beetroot subsp. maritime The RZ-3 locus is integrated into plants of the genus *Sacchariformis*, especially... Beetroot subsp. common var. very high Subsequently, markers with good diagnostic value were developed for tracing RZ-3 genomic segments in fine mapping, which proved particularly difficult because the target regions are reproducible over a wide range. Surprisingly, however, it was possible to successfully develop several diagnostic biomarkers that function only with a specific marker technology (such as pyrosequencing, i.e., as a PSQ marker) or that are zero alleles.

[0034] Despite these technical challenges, it is possible to confine the RZ-3 locus to a 0.67 cM genomic region using comprehensive analysis of these markers. This equates to a physical length of approximately 340,000 bp. Despite intensive development, it is only possible to further reduce the size of the gene using marker-assisted methods to a limited extent. Beetroot subsp. maritimeIntrogression around the gene was performed, and candidate genes for the RZ-3 gene were identified. However, from a breeding perspective, it was desirable in any case to further shorten the introgression to eliminate any potential "linkage drag" tightly coupled to the RZ-3 gene. Finally, through fine localization in many steps and incorporating sequence information from physical maps, the target region could be limited to only about 0.07 cM. However, this was only possible because a total of 8004 tests were performed, including informative recombinant BC2S1 or BS2S2 plants, with in-depth analysis of 90-180 progeny in each case. This was necessary because resistance expression is not always clear for unknown reasons. These progeny were genotyped into individual plants and phenotypically classified in parallel. Phenotypic results (homozygous resistance – RR; heterozygous resistance – Rs; homozygous susceptibility – ss) of informative recombinants were detected using statistical methods (t-test, power analysis), and conclusions were drawn regarding the genotype of informative recombinants.

[0035] Within a relatively small target region of approximately 38,000 bp, ten genes in the susceptible genotype could be annotated. Using novel markers specifically describing the target region, overlapping clones were identified and sequenced from the resistance BAC library targeting this region. Due to the repetitive nature of the target region, the sequences of the susceptible genotypes showed multiple small segments with unknown sequence contents. Assembling the RR and ss sequences was particularly arduous. However, it was possible to identify the putative resistance gene. This included (especially in almost all ss genotypes) a retrotransposon approximately 8,000 bp in length between the LRR and IR domains, which was undetectable in the RR genotype. The amino acid sequence predicted from the putative resistance gene sequence indicated that this gene likely encodes the NB-ARC-LRR protein. It can be hypothesized that the insertion of this retrotransposon disrupts the function of this gene in the susceptible ss genotype because it separates the internal repetitive domain (IR) from the other two domains (NB-ARC and LRR).

[0036] As can be inferred from Figures 1, 2, and 3, a comparison of the NBS-LRR gene in the ss genotype with that in the RR genotype also reveals diagnostic polymorphism. Based on these polymorphisms in the NBS-LRR gene, markers were developed and tested in a large group of approximately 100 ss and RR genotypes. Marker patterns, along with comparative sequencing in the target gene, confirmed that the insertion is indeed always associated with susceptibility. However, several ss genotypes were found to lack the retrotransposon insertion and remain susceptible. Nevertheless, these ss genotypes can be clearly distinguished from the RR genotypes using the markers describing the diagnostic polymorphisms in Figures 1, 2, and / or 3.

[0037] In the analyzed population, recombinants were identified in the target region showing recombination between the NBS-LRR gene and a downstream adjacent annotated putative gene (possibly encoding an ankyrin repeat sequence protein). In two plant examples, recombination between the NBS-LRR gene and an upstream adjacent annotated putative gene encoding the DUF565 protein (a protein of unknown function) was found. Resistance analysis of the progeny of all these recombinant plants (removing single genes upstream and downstream of the NBS-LRR gene) clearly demonstrated that the gene located between the ankyrin repeat gene and the DUF565 gene, specifically the NBS-LLR gene characterized in this paper, is responsible for resistance in the RR genotype. Figure 4 The physical map of the RZ-3 target area and the developed markers are displayed. Figure 5 The data shown are the genotype data of eight densely recombinant lines and the statistical analysis of their offspring.

[0038] On another front, the present invention relates to a method for identifying nucleic acid molecules encoding proteins that confer resistance to the pathogen BNYVV in beet plants expressing the protein. The method includes detecting the absence of insertions in the encoding nucleotide sequence of the nucleic acid molecule. Preferably, the method includes detecting the absence of insertions, particularly retrotransposons, in the encoding nucleotide sequence of the nucleic acid molecule. The retrotransposons can be, for example, about 500 bp, about 1000 bp, about 2000 bp, about 4000 bp, about 8000 bp, or longer than about 8000 bp. In one specific embodiment of the method, the nucleic acid molecule is the aforementioned nucleic acid molecule of the present invention and encodes the resistance-conferring RZ-3 gene or a functional homolog of RZ-3. The beet plant is preferably... Beetroot subsp. maritime or Beetroot subsp. wish garri var. very high (Sugar beet). Those skilled in the art will know what methods are suitable for detecting the absence of an insertion. For example, those skilled in the art who are familiar with the nucleic acid molecules of the present invention disclosed herein can be able to develop molecular markers to detect the presence or absence of an insertion in the aforementioned region of the NBS-LLR gene (see exemplary methods in the embodiments). The present invention includes such markers and their use for detecting the presence or absence of an insertion in order to select resistant (especially BNYVV resistant) plants, particularly... Beetroot subsp. maritime or Beetroot subsp. common var. al very(Sugar beet). This labeling preferably describes the locus located at the insertion site of the retrotransposon. The insertion site refers to the transition point between the genomic DNA and the retrotransposon on the 5' and / or 3' side of the insertion. The transition point should be broadly defined, and the labeled locus can be arranged on the DNA at distances of less than 1000 nucleotides upstream or downstream of the insertion site, preferably less than 800 or 600 nucleotides, particularly preferably less than 400, 200, 150, 50, 40, 30, 100, 20, or 10 nucleotides. Alternatively, in addition to the step of detecting the presence or absence of an insertion in the coding nucleotide sequence of the nucleic acid molecule, the method may also include using molecular markers for identifying polymorphisms (especially diagnostic polymorphisms) to detect at least one polymorphism according to Figures 1, 2, and / or 3 in the coding nucleotide sequence of the nucleic acid molecule of the present invention, preferably at least two or three polymorphisms according to Figures 1, 2, and / or 3, particularly preferably at least four, five, or more polymorphisms according to Figures 1, 2, and / or 3. Preferably, each polymorphism (especially each diagnostic polymorphism) is detected using at least one molecular marker. Those skilled in the art know which marker techniques should be applied to detect the corresponding polymorphism, and how to construct molecular markers for this purpose (literature). Furthermore, the present invention includes molecular markers describing or detecting polymorphisms according to Figures 1, 2, and / or 3, and the use of molecular markers for detecting polymorphisms according to Figures 1, 2, and / or 3. Additionally, the above-described identification method also constitutes a method for selecting plants resistant to BNYVV. The selection method includes a terminating step of selecting resistant plants.

[0039] Furthermore, it may also indicate that, within the examined RR genotype, there is a genomic DNA sequence portion according to SEQ ID NO:4 upstream and adjacent to RZ-3 (SEQ ID NO:1), and a genomic DNA sequence portion according to SEQ ID NO:5 downstream and adjacent to RZ-3 (SEQ ID NO:1), which is tightly coupled to the RZ-3 gene and is therefore a DNA region highly suitable for developing diagnostic markers for RZ-3. Therefore, the present invention relates to a method for selecting plants resistant to BNYVV. The selection method includes using molecular markers on the DNA sequence of SEQ ID NO:4 and / or the DNA sequence of SEQ ID NO:5, and a termination step for selecting resistant plants. Those skilled in the art know how to develop and use markers based on the disclosed sequences.

[0040] For the breeding and development of new resistant beet varieties, the present invention offers the following further advantages: Sequence information and the identified polymorphisms allow for the differentiation of resistance RR and susceptibility ss alleles in the disclosed genes, making it possible to develop markers directly within the genes. This constitutes a significant advantage for plant growers, particularly for developing optimized lines without linkage baggage. Furthermore, knowledge of the sequence structure can be used to identify additional resistance genes, especially those against root rot, which may be partially homologous.

[0041] The application of the resistance gene alleles disclosed in this invention in cis- or trans-genetic methods opens up the possibility of developing new resistant species of the *Sacchariformis* genus, exhibiting higher resistance based on dose-response, or where resistance interruption can be avoided and resistance expression can be optimized due to the stacking of the disclosed gene with other resistance genes. It is also conceivable to develop new resistance alleles through tilling or selective engineering modification of the gene.

[0042] This invention also relates to the application of the identified resistance RZ3 gene allele in the genetic or molecular superposition with other genetic elements, which can confer agronomically beneficial traits in plants. Therefore, the economic value of crop plants can be significantly increased, for example, by increasing yield performance or developing new planting areas where these plants could not previously be grown, especially due to biotic factors such as severe pathogen stress or abiotic factors such as drought. Agronomically beneficial traits, for example, are tolerance to herbicides such as glyphosate, glufosinate, or ALS inhibitors. Many other herbicides and their suitability are known to those skilled in the art from the prior art. Those skilled in the art can refer to the prior art to gain knowledge of which genetic elements to use and in what manner to achieve the corresponding tolerance in plants. Another example of agronomically beneficial traits is additional pathogen resistance, where pathogens can be, for example, insects, viruses, nematodes, bacteria, or fungi. For example, broad-spectrum pathogen defense in plants can be achieved by combining different pathogen resistance / tolerances, as the genetic elements may have complementary effects. For this purpose, those skilled in the art know, for example, many resistance genes as genetic elements. Another example of an agronomically beneficial trait is tolerance to low temperatures or frost. Plants with this trait can be sown earlier in the year, or, for example, can remain in the field longer, even during frost, which can lead to increased yields, for example. Here, those skilled in the art can also refer to the prior art to find suitable genetic elements. Further examples of agronomically beneficial traits are water use efficiency, nitrogen use efficiency, and harvest. Genetic elements that can be used to confer such traits can be found in the prior art.

[0043] Those skilled in the art are also aware of many modifications for pathogen defense. In addition to the frequently described R gene family, the Avr / R method, Avr gene complementation (WO2013 / 127379), R-gene self-activation (WO2006 / 128444), HIGS (host-induced gene silencing) method (e.g., WO2013 / 050024), or VIGS (virus-induced gene silencing) method can also be advantageously used. In particular, R-gene self-activation may be of great significance to this invention. For this purpose, a nucleic acid encoding a self-activated resistance protein is created, said protein being used to generate resistance to pathogens in plants. This nucleic acid then has only a limited portion of an NBS-LRR resistance gene such as the RZ3 gene, which extends downstream from the 5' end of the coding region of said NBS-LRR resistance gene to the starting point of the NBS domain of said NBS-LRR resistance gene, wherein said NBS-LRR resistance gene is not a TIR-NBS-LRR resistance gene.

[0044] Furthermore, the present invention also includes the use of the resistance RZ3 gene allele identified by the methods described above, which is used in combination with one of the above-described modifications or the above-described genetic elements that can confer one or more agronomically beneficial characteristics in plants.

[0045] Variations and embodiments of the present invention will be described by way of example with reference to the accompanying drawings and sequences: sequence: SEQ ID NO: 1 Genomic DNA sequence of the resistance gene RZ-3. This sequence includes nucleotides 1 to 1403 of the promoter regulatory region. SEQ ID NO:2 Predicted protein sequence of resistance protein RZ-3_1 SEQ ID NO:3 Predicted protein sequence of resistance protein RZ-3_2 The upstream adjacent chromosome region of SEQ ID NO:4 RZ-3 (SEQ ID NO:1) Downstream adjacent chromosome region of SEQ ID NO:5 RZ-3 (SEQ ID NO:1) Shared sequence of the RZ-3 gene genomic sequence in SEQ ID NO:6 ss genotype SEQ ID NO:7 The target sequence of the RZ3 gene in the RNAi construct of the vector pZFN-C48-RNAi. Attached Figure Description

[0046] Figure 1. AI: Comparison of nucleotide sequences between common sequences in the genomic sequences of the RZ3 gene (SEQ ID NO: 6) in the ss genotype and the RZ-3 gene (SEQ ID NO: 1) in the RR genotype. Diagnostic polymorphisms are indicated in gray and bold. Non-diagnostic polymorphisms are indicated by underlines. Potential transcription start sites of the genes are indicated by arrows. They result in two polypeptide variants, RZ-3_1 and RZ-3_2. The black triangles at the top indicate the locations of retrotransposons.

[0047] Figure 2 AL: Amino acid sequence comparison between the predicted polypeptide (RZ-3_1; SEQ ID NO:2) from the RR genotype and polypeptides from 22 different ss genotypes. Diagnostic polymorphisms are indicated in gray and bold. Non-diagnostic polymorphisms are indicated by underline.

[0048] Figure 3 AL: Amino acid sequence comparison between the predicted polypeptide (RZ-3_2; SEQ ID NO:3) from the RR genotype and polypeptides from 22 different ss genotypes. Diagnostic polymorphisms are indicated in gray and bold. Non-diagnostic polymorphisms are indicated by underline.

[0049] Figure 4 Physical map of the RZ-3 target region. Five genes are annotated in the target region of the sensitive reference genotype shown: (“2” (DUF565), “3” (proposed protein), “4” (NBS-LRR candidate gene), “5” (retrotransposon), and “6” (ankylin repeat sequence). In the sensitive reference sequence, the NBS-LRR candidate gene (“4”) contains a retrotransposon (“5”). This retrotransposon is completely absent in the resistant sequence, so only four genes can still be annotated in the resistant genotype (“2”, “3”, “4”, and “6”). The most densely recombinant positions (recombinants: 111T_3515 / ZR11007_03075 with the number “7” and 111PB3645 / ZR08093_05621 with the number “8”) are marked at the top. With their help, the shorter target region “1” can be restricted. The markers developed from recombination analysis for this purpose are reproduced as short black lines at the bottom of the figure. A gene segment ("9") selected from the domain region "10" as the target sequence was used to validate the gene for gene splicing against the resistance RZ-3 allele in the RNAi method.

[0050] Figure 5Marker analysis of the most densely packed recombinants in the RZ-3 target region (small letters in bold boxes represent computer-generated marker data). Phenotyping and genotyping were performed on 1051 progeny from 8 recombinant lines. Based on marker data in the NBS-LRR candidate genes, or if the NBS-LRR candidate genes were homozygous RR and ss, based on marker data from the splice flanking regions, the progeny were divided into three groups (RR resistance homozygotes, Rs heterozygotes, and ss sensitivity homozygotes). Furthermore, the corresponding ELISA values ​​were reproduced. Splicing and non-splicing in the progeny were examined using t-tests and Wilcoxon statistics. Based on these results, the candidate genes could be clearly defined between markers s3e5800s01 and s3e5873s01.

[0051] Figure 6 Transformation vector pZFN-C48-RNAi: d35S promoter; C48 s: sense direction of C48 sequence; AtAAP6 intron 2: Arabidopsis amino acid permease 6 intron; C48 as: antisense direction of C48 sequence; Nos-t: nos terminator; LB flanking site: left boundary flanking site; ZFN site: zinc finger nuclease recognition site (complementary); Pnos: nos promoter; NPT: coding sequence; neomycin phosphotransferase (npt) gene; pAG7: pAG7 terminator; Bvpal 3'UTR: 3' untranslated region of the beet (Beta vulgaris) Pal gene; LB: left boundary; aadA: coding sequence; aminoglycoside-3″-adenylate transferase (AAD); pVS1-REP: pVS1 origin of replication; ColE1 ori: ColE1 origin of replication; RB: right boundary. Detailed Implementation

[0052] Example: Localization and fine mapping of the RZ-3 gene / physical genetic map RZ-3 resistance (also known as C48 resistance or C48) was mapped to a genetic distance of 0.0714 cM between markers on chromosome 3 at 57.1 and 57.8 cM (internal reference map) using multiple steps of mapping and fine mapping. A total of 8004 plants from an S504 (sensitive genotype) x T74 (resistant genotype) cross were analyzed for mapping. Parallel to C48 QTL mapping, new informative markers were developed in a target-oriented manner after each mapping step to narrow down the C48 target region.

[0053] Further analysis of the progeny of the informative recombinants confirmed the fine-grained location coordinates. For this purpose, in-depth analysis was performed on plants of the informative recombinants BC2S1 or BC2S2, using 90–180 progeny of each. Genotyping and phenotyping were performed on these progeny in parallel on an individual plant basis. Statistical methods (t-test, power analysis) were used to detect the phenotypes of the informative recombinants (homozygous resistance RR / heterozygous Rs / homozygous ss), thus allowing conclusions to be drawn regarding the genotype of the informative recombinant. Since the homozygous categories (RR and ss) in the progeny differ in terms of resistance, the gene is present in the heterozygous region (Rs) of the parent plant; otherwise, it is present in the homozygous region (RR or ss) of the parent plant.

[0054] Physical maps of susceptible genotypes for root rot were generated by projecting markers and their genetic locations onto chromosome sequences. Novel informative markers were developed based on reference sequences and additional comparative sequencing of resistance genotypes (next-generation sequencing and Sanger sequencing) by restricting the C48 QTL region.

[0055] The region identified by fine mapping contained a sequence length of 37,996 base pairs within the sensitive reference sequence (the location of flanking SNP markers). Collinearity between the genetic and physical maps in the target region was consistent (12 marker sequences in the target region).

[0056] Identification and sequencing of resistant BAC clones A BAC library was developed for the selected RZ-3(C48) resistance genotype. This BAC library was sampled using markers used in the C48 QTL regions. Several BAC clones were identified for the target region. Three BAC clones of different lengths containing the complete target region were selected for sequencing. The BAC clones were sequenced and assembled "de novo" based on the resulting reads. Among the obtained resistance sequence contigs, the longest sequence was 110,909 bp (34,537 reads) and contained the complete target region.

[0057] Comparison of Sensitivity and Resistance Sequences – Sequence Evaluation Collinearity of the two SS and RR sequences was compared using different software tools. Gene annotation was performed using Maker and Pedant software for both resistant and sensitive sequences. Gene annotation on both sequences showed identical sequences to the assumed genes. However, surprisingly, significant differences could be identified in one of these genes, particularly in the gene of the present invention (RZ-3). In the sensitive genotype, a retrotransposon was annotated in the identified NBS-LRR gene. This transposon inserts into the gene between two domains, the LRR and IR domains. The resistant genotype does not have this insertion and is shown in SEQ ID NO: 1. Furthermore, the predicted polypeptide sequences were compared and evaluated (partially shown in Figures 2 and 3).

[0058] Comparative sequencing of NB-ARC-LRR candidate genes The NB-ARC-LRR candidate gene was sequenced comparatively in two steps. The retrotransposon insertion site was validated in a genotype set of 92 resistant and susceptible genotypes. This analysis showed that none of the resistant genotypes had a retrotransposon insertion. In the susceptible genotypes, the insertion was detectable in over 90%. Therefore, the detection of the insertion appeared to be associated with susceptible genotypes. However, due to the discrepancies found (approximately 10% of the remaining susceptible genotypes did not have the insertion), sequencing was extended to the entire gene prior to the insertion site (SEQ ID NO: 1) in the second step using the promoter region. In total, 31 selected resistant and susceptible genotypes, including discrepancies, were sequenced and compared. The results showed that all resistant genotypes, divided into seven distinct sources of resistance, were 100% identical across approximately 4100 base pairs compared. Furthermore, complete diagnostic polymorphisms were found in the nucleotide sequences, some of which resulted in amino acid substitutions in the protein sequence (see Figures 1, 2, and 3). Some of these substitutions, particularly in the domain regions, may lead to loss of function of the resistance protein identified in the ss genotype. Furthermore, three insertions / deletions (INDELs) completely coupled to resistance (linkage disequilibrium = 1) were also found in the promoter region (Figure 1). These insertions / deletions should also be considered potential candidates for loss of function.

[0059] Validation of the gene using dense recombinants In the analyzed population of 8004 plants, 16 recombinants were identified in the target region (a fine-mapping region of 37996 bp). Of these 16 genotypes, 9 plants contained a recombinant between the NB-ARC-LRR protein and the right-hand adjacent ankyrin repeat protein. In two plants, the recombinant was located between the NB-ARC-LRR protein and the left-hand adjacent DUF565 protein (a protein of unknown function). Analysis of the progeny of all these recombinant plants (with gene distances to the left and right sides) clearly demonstrated that the gene was located between DUF565 and the ankyrin repeat protein, and specifically demonstrated that only the NB-ARC protein is responsible for resistance.

[0060] Exemplary detection of the absence of transposon insertion To detect retrotransposon insertions, three specific master primer combinations were developed. The first and second primer combinations detect insertions because, in each case, one primer in the two primer pairs is located within the retrotransposon (to the left or right of the transposon), while the second primer binds directly before or after the transposon. The third primer pair detects the absence of a retrotransposon because the binding sites of the primers are both before and after the transposon. Then, under standard conditions, PCR products are generated only when the retrotransposon is absent; otherwise, if the retrotransposon is present, the PCT product would be too large, and no amplicons would be generated in this case.

[0061] The gene was verified using RNAi. In addition to verifying the gene using the dense recombinant method described above, the resistance effect of the gene was further investigated using RNA interference. For this purpose, a resistant standard sugar beet genotype was transformed with a DNA construct encoding a double-stranded hairpin RNA. This dsRNA enables post-transcriptional silencing of the gene, which reduces or shuts down the activity of the resistance RZ-3 gene allele, thereby converting previously resistant sugar beet genotypes into those susceptible to rhizomatous root rot.

[0062] To provide a suitable DNA construct, a well-defined target sequence region (SEQ ID NO:7) of 434 base pairs long was selected from the allele of the resistance RZ3 gene; Figure 4 The vector pZFN was amplified by PCR and cloned into the vector in both sense and antisense directions, making it suitable for the synthesis of hairpin structures. Figure 6The vector possesses a double CaMV 35S promoter, a multiple cloning site / intron from the gene AtAAP6 (encoding Arabidopsis amino acid permease), additional multiple cloning sites, and a nos terminator. Sugar beet was converted using the provided vector according to the experimental method of Lindsey & Gallois (1990), with kanamycin as the selection marker. After several selection steps, successful transformation was checked on transgenic seedlings by PCR, detecting the presence of the nptII gene, the AAP6 intron, and the two t-DNA boundary sequences (LB / RB). man The absence of [a specific nutrient] was observed. Positive seedlings were each cloned in vitro to 30 seedlings, rooted, and then transferred to greenhouse soil. Approximately two weeks later, the transgenic sugar beet plants were planted in root rot-contaminated soil and cultured there for 8 to 10 weeks. As a control, non-transformed plants were transformed under the same conditions using the same resistance genetic standard. To detect the spread of root rot, stems of the sugar beet plants were harvested and quantified using an ELISA test against BNYVV challenge, where low ELISA values ​​indicated resistance and high values ​​indicated sensitivity (Mechelke 1997, Clark & ​​Adams 1977). The mean ELISA value of the transformed sugar beet was 3.55, significantly higher than the control (which was also resistant, with a mean of 1.27), and matched the sensitivity standard D108_ss (Table 1). The results of the ELISA test accordingly indicated that previously resistant plants were susceptible to BNYVV due to specific gene silencing of the resistance RZ-3 allele in the transformed background. Therefore, the gene of this invention can be clearly identified as the resistance gene RZ3.

[0063] Table 1: ELISA test results after statistical analysis (D108_ss = sensitivity standard; 6921_RR = resistance transformation background; 6921_RNAi = resistance transformation background with dsRNA targeting the RZ3 gene).

[0064] References Clark, MF; Adams, AN (1977): Characteristics of the microplatemethod of enzyme-linked immunosorbent assay for the detection of plantviruses. J. Gen. Virol. 34, 475–483 Depicker A, Stachel S, Dhaese P, Zambryski P, Goodman HM (1982)Nopaline synthase: transcript mapping and DNA sequence. J Mol Appl Genet. 1(6): 561-73. Esser K (2000) Kryptogamen 1: Cyanobakterien Algen Pilze FlechtenPraktikum und Lehrbuch. Springer Publishing House, Berlin, Heidelberg,3 rd edition. 2000. Gidner S, Lennefors BL, Nilsson NO, Bensefelt J, Johansson E,Gyllenspetz U, Kraft T (2005) QTL mapping of BNYVV resistance from the WB41source in sugar beet. Genome 48: 279-285. Larson RL, Wintermantel WM, Hill A, Fortis L, Nunez A (2008) Proteomechanges in sugar beet in response to Beet necrotic yellow vein virus.Physiological and Mol. PI. Pathol. 72: 62-72. Lindsey, K., and P. Gallois (1990) "Transformation of sugar beet(Beta vulgaris) by Agrobacterium tumefaciens." Journal of experimental botany 41.5: 529-536. Martin GB, Bogdanove AJ; Sessa G (2003) Understanding the functions of plant disease resistance proteins. Annual Review of Plant Biology 54: 23-61. Mechelke W (1997) Problems in rhizomania resistance breeding, Lectures for plant breeding, resistance breeding in sugar beets, Society for Plant Breeding eV, 113-123. Odell JT, Nagy F, Chua NH (1985) Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313,810 - 812 Rushton PJ, Torres JT, Parniske M, Wernert P, Hahlbrock K, andSomssich IE (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. Sambrook J, Russell DW (2001) Molecular cloning. A laboratory manual, Cold Spring Harbor Laboratory Press, 3rd ed. 2001. Schmidlin LEDEB, Weyens G, Lefebvre M, Gilmer D (2008) Identificationof differentially expressed root genes upon rhizomania disease. Mol. PlantPathol. 9(6):741-51. Scholten OE, Bock TSMD, Klein-Lankhorst RM, Lange W (1999)Inheritance of resistance to Beet necrotic yellow vein virus in Beta vulgarisconferred by a second gene for resistance. Theor. Appl. Genet. 99:740-746. Sohi HH, Maleki M(2004) Evidence for presence of types A and B ofbeet necrotic yellow vein virus (BNYVV) in Iran. Virus Genes 29(3): 353-8. Van Ooijen G, Mayr G, Kasiem MMA, Albrecht M, Cornelissen BJC, TakkenFLW (2008) Structure–function analysis of the NB-ARC domain of plant diseaseresistance proteins. Journal of Experimental Botany, 59(6): 1383-1397 WO / 2000 / 29592 (Max-Planck-Gesellschaft zur Förderung derWissenschaften e.V.). Chimeric promoters capable of mediating gene expressionin plants upon pathogen infection and uses thereof. WO / 2006 / 128444 (KWS SAAT AG). AUTOACTIVATED RESISTANCE PROTEIN. WO / 2007 / 147395 (KWS SAAT AG). Pathogen induzierbarer synthetischerPromotor. WO / 2013 / 127379 (KWS SAAT AG). PATHOGEN-RESISTANT TRANSGENIC PLANT. WO / 2013 / 050024 (KWS SAAT AG). TRANSGENIC PLANT OF THE SPECIES BETAVULGARIS HAVING ENHANCED RESISTANCE TO CERCOSPORA. WO / 2013 / 091612 (KWS SAAT AG). NOVEL PLANT-DERIVED CIS-REGULATORYELEMENTS FOR THE DEVELOPMENT OF PATHOGEN-RESPONSIVE CHIMERIC PROMOTORS.

Claims

1. A nucleic acid molecule encoding a polypeptide, said polypeptide being capable of conferring resistance to pathogens in plants expressing the polypeptide, characterized in that... The nucleic acid molecule contains nucleotide sequences selected from the following: a) A nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3; b) The nucleotide sequence containing the coding sequence of the DNA sequence of SEQ ID NO: 1; c) A nucleotide sequence that hybridizes with the complementary sequence of the nucleotide sequence of a) or b) under stringent conditions; d) A nucleotide sequence encoding a polypeptide, said polypeptide being derived from the polypeptide encoded by the nucleotide sequence of a) or b) by substitution, deletion and / or addition of one or more amino acids in the amino acid sequence encoded by the nucleotide sequence of a). e) A nucleotide sequence encoding a polypeptide having at least 60% identical amino acid sequence to the amino acid sequence encoded by the nucleotide sequence of a) or b); f) A nucleotide sequence encoding at least one nucleotide-binding domain (NBS) corresponding to amino acid positions 168-227 of SEQ ID NO: 2 or amino acid positions 182-241 of SEQ ID NO: 3, at least one leucine-rich domain (LRR) corresponding to amino acid positions 591-613 of SEQ ID NO: 2 or amino acid positions 605-627 of SEQ ID NO: 3, and / or at least one internal repeating domain (IR) corresponding to amino acid positions 1013-1072 of SEQ ID NO: 2 or amino acid positions 1027-1086 of SEQ ID NO:

3.

2. A vector comprising the nucleic acid molecule of claim 1.

3. A host cell comprising the nucleic acid molecule of claim 1 or the vector of claim 2.

4. A polypeptide that confers resistance to pathogens in plants expressing the polypeptide, and is encoded by the nucleic acid molecule of claim 1.

5. A transgenic plant cell comprising the nucleic acid molecule of claim 1 as a transgene, or comprising the vector of claim 2.

6. A transgenic plant or a portion thereof, comprising the plant cells of claim 5.

7. The seed of the plant of claim 6, wherein the seed contains the nucleic acid molecule of claim 1 as a transgene.

8. A method for producing transgenic plant cells, characterized in that, The method includes the step of introducing the nucleic acid molecule of claim 1 or the vector of claim 2 into plant cells.

9. A method for producing transgenic plants, characterized in that... The method includes the following steps: a) Introducing the nucleic acid molecule of claim 1 or the vector of claim 2 into plant cells, and b) Regenerate transgenic plants from the transgenic plant cells of step a).

10. A promoter regulatory sequence, said promoter controlling the expression of a gene comprising the nucleic acid molecule of the present invention, characterized in that... The regulatory sequence can confer or regulate the expression of a heterologous DNA sequence during pathogen infection, and the regulatory sequence comprises a nucleic acid molecule having a nucleotide sequence of nucleotides 1-1403 of SEQ ID NO:

1.

11. A recombinant DNA molecule comprising the regulatory sequence of claim 10.

12. A host cell transformed with the regulatory sequence of claim 10 or with the recombinant DNA molecule of claim 11.

13. A transgenic plant, plant tissue or plant cell comprising the regulatory sequence of claim 10 or the recombinant DNA molecule of claim 11 as a transgenic material.

14. A method for identifying nucleic acid molecules encoding proteins, said proteins being capable of conferring resistance to the pathogen BNYVV in beet plants expressing the protein, characterized in that... The method includes the following steps: i. Detection confirms that the coding nucleotide sequence of the nucleic acid molecule of claim 1 does not contain an insertion, or ii. Detecting at least one polymorphism in the encoding nucleotide sequence of the nucleic acid molecule of claim 1 according to Figures 1, 2 and / or 3, using a molecular marker to identify said polymorphism.

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