# US 2021/0340557 A1 · Beet necrotic yellow vein virus (bnyvv)-resistance modifying gene

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- Number: US 2021/0340557 A1
- Document ID: US20210340557A1
- Status: Published application
- Kind: A1
- Application number: 17/274,692
- Filed: 2019-09-11
- Published: 2021-11-04
- Earliest claimed: 2018-09-11
- Assignees: KWS SAAT SE & Co. KGaA
- Applicants: KWS SAAT SE & Co. KGaA
- Inventors: Ann-Katrin MÜNNEKHOFF; Britta SCHULZ; Daniel Fabian STIRNWEIS; Ottó TÖRJÉK; Dietrich BORCHARDT
- Web page: https://patentgrep.com/patent/US20210340557A1

## CPC

- C12N 15/8283: {for virus resistance}
- C07K 14/415: from plants
- C12Q 1/6895: for plants, fungi or algae
- C12Q 2600/13: Plant traits
- C12Q 2600/156: Polymorphic or mutational markers
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

A method for establishing, restoring or increasing the Rz2 gene mediated resistance to the Beet necrotic yellow vein virus (BNYVV) in the target plant is enabled via the identification and provision of a gene which modulates said resistance according to the invention. In particular, the BNYVV resistance mediated by the Rz2 resistance gene depends on the presence of the gene of the present invention. The gene being involved in conferring said resistance, and embodiments of the present invention that are described in the preceding, offer additional applications, e.g., the use of the resistance modifying gene allele in cis-genetic or trans-genetic approaches, with the goal of developing new resistant cultivars.

## Claims

### Claim 1

A nucleic acid molecule encoding a polypeptide which is involved in resistance against Beet necrotic yellow vein virus (BNYVV) mediated by the Rz2 resistance gene in a plant in which the polypeptide is expressed, wherein the nucleic acid molecule comprises a nucleotide sequence which is selected from the group consisting of:
(a) a nucleotide sequence that encodes a polypeptide having the amino acid sequence according to SEQ ID No. 45;
(b) a nucleotide sequence that comprises the coding DNA sequence according to SEQ ID No. 44
(c) a nucleotide sequence that hybridizes with a complementary sequence of the nucleotide sequence according to (a) or (b) under stringent conditions;
(d) a nucleotide sequence that encodes a polypeptide which, via substitution, deletion, and/or addition of one or more amino acids of the amino acid sequence, differs from a polypeptide that is encoded by the nucleotide sequence according to (a) or (b);
(e) a nucleotide sequence that encodes a polypeptide which has an amino acid sequence that is at least 70% identical to the amino acid sequence according to SEQ ID No. 45; and
(f) a nucleotide sequence that comprises the coding DNA sequence which is at least 70% identical to the DNA sequence according to SEQ ID No. 44,
wherein the amino acid sequence of the encoded polypeptide has a proline (P) at position 25 with reference to SEQ ID No. 45, a proline (P) at position 72 with reference to SEQ ID No. 45 or a valine (V) at position 186 with reference to SEQ ID No. 45, or
wherein the nucleic acid of the coding DNA sequence has a cytosine (c) at position 73 with reference to SEQ ID No. 44, a cytosine (c) at position 214 with reference to SEQ ID No. 44, or a guanine (g) at position 556 with reference to SEQ ID No. 44, preferably
wherein the polypeptide has the amino acid sequence of SEQ ID No. 8, 12, 16, or 20, or the nucleotide sequence that encodes said polypeptide is SEQ ID No. 5, 6, 7, 9, 10, 11, 13, 14, 15, 17, 18 or 19, or the coding DNA sequence is a nucleotide sequence according to SEQ ID No. 7, 11, 15, or 19.

### Claim 2 (depends on 1)

A nucleic acid molecule which specifically hybridizes to the nucleic acid molecule according to claim 1 and encodes a non-functional allele of the polypeptide, which interferes with resistance against BNYVV mediated by the Rz2 resistance gene in a plant in which the polypeptide is expressed, wherein the nucleic acid molecule preferably comprises a nucleotide sequence which is selected from
(a) a nucleotide sequence that encodes a polypeptide having an amino acid sequence according to SEQ ID No. 4 or a polypeptide having an amino acid sequence that is at least 70% identical to SEQ ID No. 4, wherein the polypeptide comprises an amino acid sequence having at least one amino acid substitution due to one or more mutations in the nucleotide sequence, preferably wherein proline (P) at position 25 with reference to SEQ ID No. 45, proline (P) at position 72 with reference to SEQ ID No. 45, and/or valine (V) at position 186 with reference to SEQ ID No. 45 is substituted with another amino acid, preferably wherein proline (P) at position 25 is substituted with alanine (A), proline (P) at position 72 is substituted with threonine (T), and/or valine (V) at position 186 is substituted with isoleucine (I); and/or
(b) a nucleotide sequence that comprises the coding DNA sequence according to SEQ ID No. 3, that comprises a coding DNA sequence which is at least 70% identical to the DNA sequence according to SEQ ID No. 3, or that hybridizes with a complementary sequence of SEQ ID No. 3 under stringent conditions, wherein the nucleotide sequence comprises at least one nucleic acid substitution due to at least one mutation leading to an amino acid substitution, preferably wherein one or more nucleotides are substituted at positions 73-75 with reference to SEQ ID No. 44, at positions 214-216 with reference to SEQ ID No. 44 and/or at positions 556-558 with reference to SEQ ID No. 44, preferably wherein cytosine (c) at position 73 is substituted with guanine (g), cytosine (c) at position 214 is substituted with adenine (a), and/or guanine (g) at position 556 is substituted with adenine (a).

### Claim 3 (depends on 1)

A polypeptide which is encoded by the nucleic acid molecule according to claim 1.

### Claim 4 (depends on 1)

A vector or expression cassette which comprises the nucleic acid molecule according to claim 1, preferably wherein the nucleic acid molecule is operably linked to a heterologous regulatory element.

### Claim 5 (depends on 1)

A cell which comprises the nucleic acid molecule according to claim 1.

### Claim 6 (depends on 1)

A plant or a portion thereof comprising the nucleic acid molecule according to claim 1 endogenously or transgenically as transgene, preferably wherein the plant additionally comprises the Rz2 resistance gene mediating BNYVV resistance endogenously or transgenically.

### Claim 7 (depends on 6)

Seed or a descendant of the plant according to claim 6, wherein the seed has been technically treated, whereby the technical treatment is selected from the group consisting of:
(a) Polishing,
(b) Dressing, preferably pelleting,
(c) Incrustation, and
(d) Colouring.

### Claim 8 (depends on 1)

Oligonucleotide or a pair of oligonucleotides of at least 15, 16, 17, 18, 19, or 20, preferably at least 21, 22, 23, 24, or 25, particularly preferably at least 30, 35, 40, 45, or 50, and particularly preferably at least 100, 200, 300, or 500 nucleotides in length, wherein the oligonucleotide or the pair of oligonucleotides
(i) specifically hybridizes to a nucleotide sequence as defined in claim 1; and/or
(ii) specifically hybridizes to a region in the Beta vulgaris genome that, in Beta vulgaris, cosegregates with the nucleic acid molecule according to claim 1 and/or with the nucleic acid molecule according to claim 1 together with the Rz2 gene,
preferably wherein the oligonucleotide or pair of oligonucleotides is selected from the group consisting of SEQ ID NO: 24 to 40.

### Claim 9 (depends on 1)

A method for conferring, restoring or increasing the resistance to BNYVV in a plant of the species Beta vulgaris comprising the Rz2 resistance gene mediating BNYVV resistance endogenously or transgenically, including the following steps:
(i) integration or introgression of the nucleic acid molecule according to claim 1 by means of homology-directed repair or homologous recombination—preferably, promoted by site-directed nuclease—into the genome of at least one cell of a plant of the species Beta vulgaris, and optional regeneration of a plant from the plant cell; or
(ii) increasing the expression of the nucleic acid molecule according to claim 1 in the plant—preferably, via modification of the native promoter or via fusion of the nucleic acid molecule with a heterologous promoter that exhibits a higher activity in comparison to the native promoter—in particular, after BNYVV infection; or
(iii) increase in the activity and/or stability of the functional allele of the polypeptide which is encoded by the nucleic acid molecule according to claim 1 via modification of the nucleotide sequence according to claim 1;
(iv) transformation of a plant cell with the nucleic acid molecule according to claim 1, and optionally regeneration of a transgenic plant from the transformed plant cell; or
(v) and screening/selecting for plants or plant cells, respectively, in which a functional allele of the polypeptide encoded by the nucleic acid molecule according to claim 1 has been restored and/or the expression of the non-functional polypeptide has been abolished.

### Claim 10 (depends on 1)

A method for producing a BNYVV-resistant plant comprising the nucleic acid molecule according to claim 1, including the following steps:
(I) provision of a plant of the genus Beta or a plant cell of the genus Beta comprising the Rz2 resistance gene mediating BNYVV resistance endogenously or transgenically; and
(IIa) transformation of the plant cell of (I) with the nucleic acid molecule according to claim 1; and
(IIb) regeneration of a transgenic plant from the transformed plant cell; or
(IIIa) introduction of a site-directed nuclease or nickase and optionally a repair matrix, or a site-directed base editor into the plant cell, wherein the site-directed nuclease is able to generate at least one single-strand break of the DNA or at least one double-strand break of the DNA at a predetermined location in the genome of the cell and the repair matrix comprises the nucleic acid molecule according to claim 1, or wherein the site-directed base editor is able to generate at least one single-strand break of the DNA at a predetermined location in the genome of the cell and to convert at least one nucleobase; and
(IIIb) optionally, cultivation of the cell from (IIIa) under conditions that allow modification of the genome at the predetermined location, selected from
a) a replacement of at least one nucleotide;
b) a deletion of at least one nucleotide;
c) an insertion of at least one nucleotide;
d) any combination of a)-c),
optionally by homology-directed repair or homologous recombination,
wherein the nucleic acid molecule is integrated into the genome of the plant; and
(IIIc) regeneration of a plant from the cell modified in (IIIb);
preferably wherein the predetermined location is in the nucleic acid molecule that specifically hybridizes to the nucleic acid molecule according to claim 1 and encodes a non-functional allele of the polypeptide or a position which is at most 10.000 base pairs upstream or downstream away from the nucleic acid molecule that specifically hybridizes to the nucleic acid molecule according to claim 1 and encodes a non-functional allele of the polypeptide; or
(IVa) crossing of the plant of (I) with a plant; and
(IVb) identifying and selecting a plant comprising the Rz2 resistance gene mediating BNYVV endogenously or transgenically and the nucleic acid molecule according to claim 1 endogenously or transgenically, wherein the Rz2 resistance gene and/or the nucleic acid molecule according to claim 1 is present homozygously in the genome of the plant.

### Claim 11 (depends on 1)

A method for identifying a plant of the species Beta vulgaris, a portion thereof or a seed thereof comprising the nucleic acid molecule according to claim 1, wherein the plant is capable of exhibiting resistance against BNYVV mediated by Rz2, if present in the genome of the plant, comprising:
(i) detection of the presence and/or expression of the nucleic acid molecule according to claim 1, or the presence of the polypeptide which is encoded by the nucleic acid molecule according to claim 1 in the plant, the portion thereof or the seed thereof; and/or
(ii) detection of at least one marker locus in the nucleotide sequence of the nucleic acid molecule according to claim 1 or in one or more cosegregating regions thereof, preferably wherein the detection of the at least one marker locus in the nucleotide sequence of the nucleic acid molecule according to claim 1 comprises the use of an oligonucleotide or a pair of oligonucleotides, and wherein the one or more cosegregating regions are genomic intervals in Beta vulgaris on chromosome 3 which comprise and are flanked by marker loci detectable by means of
(a) the markers s3e5985s01 (SEQ ID No. 33) and s3p4348s01 (SEQ ID No. 27), and/or
(b) the markers s3p4351s01 (SEQ ID No. 29) and s3e2247xxx (SEQ ID No. 40); and
(iii) identification and optionally selection of the plant of the species Beta vulgaris, the portion thereof or the seed thereof comprising the nucleic acid molecule according to claim 1, which is capable of exhibiting resistance against BNYVV mediated by Rz2, if present in the genome of the plant, based on the detection of (i) and/or (ii).

### Claim 12 (depends on 2)

A method for identifying a plant of the species Beta vulgaris, a portion thereof or a seed thereof comprising the nucleic acid molecule according to claim 2, comprising:
(i) detection of the presence and/or expression of the nucleic acid molecule according to claim 2, or the presence of the polypeptide which is encoded by the nucleic acid molecule according to claim 2 in the plant, the portion thereof or the seed thereof; and/or
(ii) detection of at least one marker locus in the nucleotide sequence of the nucleic acid molecule according to claim 2 or in one or more cosegregating regions thereof, preferably wherein the detection of the at least one marker locus in the nucleotide sequence of the nucleic acid molecule according to claim 2 comprises the use of an oligonucleotide or a pair of oligonucleotides, and wherein the one or more cosegregating regions are genomic intervals in Beta vulgaris on chromosome 3 which comprise and are flanked by marker loci detectable by means of
(a) the markers s3e5985s01 (SEQ ID No. 33) and s3p4348s01 (SEQ ID No. 27), and/or
(b) the markers s3p4351s01 (SEQ ID No. 29) and s3e2247xxx (SEQ ID No. 40); and
(iii) identification and optionally selection of the plant of the species Beta vulgaris, the portion thereof or the seed thereof comprising the nucleic acid molecule according to claim 2, based on the detection of (i) and/or (ii),

### Claim 13 (depends on 11)

The method according to claim 11 additionally comprising
(A) detection of the presence and/or expression of the Rz2 gene or the presence of the polypeptide encoded by the Rz2 gene in the plant, the portion thereof or the seed thereof; and/or
(B) detection of at least one marker locus in the Rz2 gene or in one or more regions cosegregating with the Rz2 gene, preferably cosegregating with a chromosomal interval comprising the nucleic acid molecule according to claim 11 and the Rz2 gene;
wherein in (iii) of claim 11 the identified and optionally selected plant, the portion thereof or the seed thereof comprises the nucleic acid molecule according to claim 11, and the Rz2 gene, based on the detection of (i) and/or (ii) of claim 11 and detection of (B).

### Claim 14 (depends on 6, 7)

A method for cultivation of plants of the species Beta vulgaris, including
(i) planting seedlings of the plant according to claim 6 or sowing seeds according to claim 7, and
(ii) grown the plants from the seedlings or seeds, and
(iii) optionally, harvest root beets from the grown plants;
wherein the method counteracts an infestation of the cultivated plants with BNYVV.

### Claim 15 (depends on 1)

A method of using the nucleic acid molecule according to claim 1 for the production of BNYVV-resistant plants, preferably of plants of the species Beta vulgaris, more preferably of plants of Beta vulgaris ssp. vulgaris var. vulgaris, Beta vulgaris ssp. vulgaris var. conditiva, oder Beta vulgaris ssp. vulgaris var. crassa/alba.