# US 9,932,603 B2 · P0 gene silencing constructs and use

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- Number: US 9,932,603 B2
- Document ID: US9932603B2
- Status: Granted
- Kind: B2
- Application number: 14/128,962
- Filed: 2012-06-15
- Published: 2014-10-23
- Granted: 2018-04-03
- Earliest claimed: 2011-06-23
- Assignees: SESVanderHave NV
- Applicants: Elodie Klein; Véronique Graff; David Gilmer; Véronique Brault; Guy Weyens; Marc Lefebvre
- Inventors: Elodie Klein; Véronique Graff; David Gilmer; Véronique Brault; Guy Weyens; Marc Lefebvre
- Web page: https://patentgrep.com/patent/US9932603B2

## CPC

- C12N 15/8283: {for virus resistance}
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/1131: {against viruses}
- C12N 15/8218: {Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention is related to a method for conveying viral resistance or tolerance to one or more virus(es), in particular to beet mild yellowing virus (BMYV) and to beet necrotic yellow vein virus (BNYVV) or to BMYV alone in a plant, in particular in a sugar beet plant. Furthermore, the present invention relates to the virus resistant or -tolerant plant obtained according to this method, as well as to seeds and progeny derived therefrom.

## Claims

### Claim 1

An RNA construct consisting of a sense segment sequence and an antisense segment sequence having sequences deduced from P0 gene of Beet Mild Yellowing Virus (BMYV) genome, wherein said sense segment and said antisense segment sequences both comprise a nucleotide fragment of a nucleotide sequence having at least 95% sequence identity with the P0 gene as set forth in SEQ ID NO: 17 from BMYV genome, wherein the fragment comprises more than 25 nucleotides and wherein the segment has a size lower than 1000 nucleotides, wherein the RNA construct is for silencing the expression of the P0 protein from BMYV.

### Claim 2 (depends on 1)

The RNA construct of claim 1, wherein the sense segment and/or antisense segment sequences further comprise a nucleotide fragment having at least 95% sequence identity with the 5′-end untranslated sequence (5′ UTR) adjacent to the P0 gene nucleotide sequence from the BMYV genome, as set forth in SEQ ID NO: 1.

### Claim 3 (depends on 1)

The RNA construct of claim 1, wherein the sense segment and antisense segment sequences further comprise a nucleotide fragment having at least 95% sequence identity with P1 gene of BMYV genome as set forth in SEQ ID NO: 4.

### Claim 4 (depends on 1)

The RNA construct according to claim 1, wherein the sense segment consists of the sequence SEQ ID NO: 1 and/or the antisense segment consists of the sequence SEQ ID NO: 3.

### Claim 5 (depends on 1)

The RNA construct according to claim 1 wherein the sense segment and antisense segment sequences further both comprise a nucleotide fragment having at least 95% sequence identity with the Beet Necrotic Yellow Vein Virus (BNYVV) genome and wherein the sense segment comprises a fragment of SEQ ID NO: 5 and the antisense segment comprises a fragment of SEQ ID NO: 6.

### Claim 6 (depends on 1)

The construct according to claim 1, wherein the sense and antisense segments comprise more than 30 nucleotides.

### Claim 7 (depends on 1)

The construct according to claim 1, wherein the sense and antisense fragments comprise between 25 and 50 nucleotides.

### Claim 8 (depends on 1)

A DNA construct transcriptable into the RNA construct according to claim 1.

### Claim 9 (depends on 8)

A vector comprising the DNA construct according to claim 8.

### Claim 10 (depends on 8, 9)

A double stranded self-complementary RNA molecule expressed by the DNA construct of claim 8, or the vector according to claim 9.

### Claim 11 (depends on 9)

A method for inducing tolerance or resistance, to at least the BMYV virus in a plant or a plant cell, the method comprising the steps of: preparing the vector according to claim 9, operably linked to one or more regulatory sequences active in the plant or the plant cell, and transforming the plant cell with the nucleic acid construct, thereby inducing resistance to at least the BMYV virus in the plant or in the plant cell.

### Claim 12 (depends on 11)

The method according to the claim 11, wherein the method further comprises inducing tolerance or resistance to another virus selected from the group consisting of the Turnip yellows virus, Curcubit aphid-borne yellows virus, Potato leafroll virus, Sugarcane yellow leaf virus, Pea Enation Mosaic Virus, Beet western yellows virus—USA, Beet chlorosis virus, Cereal yellow dwarf virus and BNYVV virus.

### Claim 13 (depends on 9)

A transgenic plant or a transgenic plant cell tolerant or resistant, to at least the BMYV virus and comprising the vector according to claim 9.

### Claim 14 (depends on 13)

The transgenic plant or transgenic plant cell of claim 13, wherein the transgenic plant is or transgenic plant cell is a sugar beet plant or cell.

### Claim 15

An RNA construct consisting of a sense segment sequence and an antisense segment sequence having sequences deduced from P0 gene of Beet Mild Yellowing Virus (BMYV) genome, wherein said sense segment and said antisense segment sequences both comprise a nucleotide fragment having at least 95% sequence identity with the P0 gene as set forth in SEQ ID NO:17 from BMYV genome, wherein the fragment comprises more than 25 nucleotides and wherein the segment has a size lower than 1000 nucleotides, wherein the RNA construct is for silencing the expression of the P0 protein from BMYV.

### Claim 16 (depends on 1)

The RNA construct of claim 1, wherein the fragments have 100% identity with the P0 gene as set forth in SEQ ID NO: 17 from BMYV genome.

### Claim 17 (depends on 1)

The RNA construct of claim 1, wherein the fragment comprises more than 50 nucleotides and wherein the segment has a size lower than 1000 nucleotides.