# US 8,569,578 B1 · Generating transgenic potatoes with novel resistance to potato cyst nematodes by silencing nematode parasitism genes of CLE -1 and CLE-4s

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- Number: US 8,569,578 B1
- Document ID: US8569578B1
- Status: Granted
- Kind: B1
- Application number: 12/847,053
- Filed: 2010-07-30
- Granted: 2013-10-29
- Earliest claimed: 2009-07-31
- Assignees: The United States of America as represented by the Secretary of Agriculture
- Applicants: Xiaohong Wang; Shunwen Lu
- Inventors: Xiaohong Wang; Shunwen Lu
- Web page: https://patentgrep.com/patent/US8569578B1

## CPC

- C07K 14/415: from plants
- 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 15/8218: {Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]}
- C12N 15/8285: {for nematode resistance}
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/531: Stem-loop; Hairpin
- Y02A 40/146: Genetically Modified [GMO] plants, e.g. transgenic plants
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Plant CLAVATA3/ESR-related (CLE) peptides have diverse roles in plant growth and development. We have isolated and characterized the function of five new CLE genes from the potato cyst nematode Globodera rostochiensis. Unlike typical plant CLEs that contain a single CLE motif, four of the five Gr-CLE genes encode CLE proteins with multiple CLE motifs. These Gr-CLEs were found to be specifically expressed within the dorsal esophageal gland cell of nematode parasitic stages, suggesting a role for their encoded proteins in plant parasitism. Overexpression of Gr-CLEs in Arabidopsis mimicked overexpression of plant CLEs and Gr-CLE proteins could rescue the Arabidopsis clv3-2 mutant phenotype when expressed within meristems. A short root phenotype was observed when synthetic GrCLE peptides were exogenously applied to roots of Arabidopsis or potato similar to the overexpression of Gr-CLEs in Arabidopsis and potato hairy roots. These results reveal that G. rostochiensis CLEs with either single or multiple CLE motifs function similarly to plant CLEs and that CLE signaling components are conserved in both Arabidopsis and potato roots. Transgenic potato hairy roots expressing Gr-CLE-1 or Gr-CLE-4 dsRNA were generated. There was an approximately 50% reduction in the average number of cysts per root in the Gr-CLE-1 or Gr-CLE-4 dsRNA transgenic lines when compared with the infected control lines, indicating that silencing nematode CLE genes through host-derived RNAi may generate novel resistance against potato cyst nematodes in transgenic potatoes.

## Claims

### Claim 1

An isolated polynucleotide that produces a double stranded ribonucleotide hairpin, said polynucleotide consisting of a fragment of at least 21 contiguous nucleotides of a nucleic acid which encodes SEQ ID NO:23, and a reverse complement of said fragment; wherein uptake by a Globodera rostochiensis nematode of the double stranded ribonucleotide hairpin inhibits the growth of said nematode.

### Claim 2 (depends on 1)

An isolated double stranded ribonucleotide molecule produced from the expression of the polynucleotide according to claim 1, wherein the taking up of said double stranded ribonucleotide molecule by a G. rostochiensis parasitic nematode inhibits the growth of said nematode.

### Claim 3

An isolated double stranded ribonucleotide molecule, defined as produced by preparing a recombinant polynucleotide molecule comprising a first and a second polynucleotide sequence, wherein the first polynucleotide sequence consists of an isolated polynucleotide molecule which encodes SEQ ID NO:23, wherein the second polynucleotide sequence is substantially the reverse complement of the first polynucleotide sequence such that the first and the second polynucleotide sequences hybridize when the recombinant polynucleotide molecule is transcribed into a ribonucleic acid molecule that forms the double stranded ribonucleotide molecule, wherein the double stranded ribonucleotide molecule inhibits the growth of a G. rostochiensis nematode.

### Claim 4

A plant transformation vector comprising a polynucleotide molecule that produces a double stranded ribonucleotide molecule, said polynucleotide molecule consisting of a fragment of at least 21 contiguous nucleotides of a nucleic acid which encodes SEQ ID NO:23, and a reverse complement of said fragment; wherein uptake by a G. rostochiensis nematode of the double stranded ribonucleotide molecule inhibits the growth of said nematode, and wherein the polynucleotide molecule is operably linked to a heterologous promoter functional in a plant cell.

### Claim 5 (depends on 1)

A transgenic plant transformed with the polynucleotide of claim 1 operably linked to a heterologous promoter functional in a plant cell.

### Claim 6 (depends on 5)

The transgenic plant of claim 5, wherein the polynucleotide is expressed in an amount effective to reduce, inhibit, or prevent nematode disease caused by G. rostochiensis compared to a control plant.

### Claim 7

A method for controlling a G. rostochiensis parasitic nematode population, comprising: providing to a G. rostochiensis nematode an agent comprising a double stranded ribonucleotide molecule that functions upon being taken up by the nematode to inhibit a biological function within said nematode, wherein the double stranded ribonucleotide molecule is transcribed from an isolated polynucleotide consisting of a fragment of at least 21 contiguous nucleotides of a nucleic acid which encodes SEQ ID NO:23, and a reverse complement of said fragment; wherein uptake by a G. rostochiensis nematode of the double stranded ribonucleotide molecule inhibits the growth of said nematode.