# US 2014/0157455 A1 · Gall wasp control agents

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- Number: US 2014/0157455 A1
- Document ID: US20140157455A1
- Status: Published application
- Kind: A1
- Application number: 14/008,914
- Filed: 2012-03-30
- Published: 2014-06-05
- Earliest claimed: 2011-03-30
- Assignees: Futuragene Israel Ltd.
- Inventors: Dror Avisar; Hanan Stein; Ziv Shani; Daniel Siegel
- Web page: https://patentgrep.com/patent/US20140157455A1

## CPC

- C12N 15/8286: {for insect resistance}
- C07K 14/43568: {from wasps}
- 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]}
- Y02A 40/146: Genetically Modified [GMO] plants, e.g. transgenic plants
- Y02A 90/40: Monitoring or fighting invasive species
- 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)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present invention relates to the field of RNA-mediated gene silencing in insect species. The present invention is based, in part, on the inventors' sequencing of genes from eucalyptus invasive species gall wasp pests Leptocybe invasa (Li) and Ophelimus maskelli (Om). In certain aspects, the invention provides Li and Om nucleic acids, derivatives thereof and the use of such nucleic acids and derivatives as gall wasp control agents.

## Claims

### Claim 1

An isolated nucleic acid comprising a sequence that selectively hybridizes under high stringency hybridization conditions to the complement of a sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 45-123, SEQ ID NO: 127-132, SEQ ID NO: 234-244.

### Claim 2 (depends on 1)

The isolated nucleic acid of claim 1 wherein said nucleic acid is 90-99.99 percent identical to said sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 45-123, SEQ ID NO: 127-132, SEQ ID NO: 234-244.

### Claim 3

An isolated nucleic acid comprising at least 17 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 45-123, SEQ ID NO: 127-132, SEQ ID NO: 234-244.

### Claim 4 (depends on 3)

The isolated nucleic acid of claim 3 wherein said nucleic acid comprises at least 25 contiguous nucleotides of sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 150-222, SEQ ID NO: 234-244.

### Claim 5 (depends on 3)

The isolated nucleic acid according to claim 3 wherein said nucleic acid is less than about 80% identical to the honey bee ortholog of said nucleic acid.

### Claim 6 (depends on 3)

A vector comprising the isolated nucleic acid of claim 3 operably linked to an expression control sequence.

### Claim 7 (depends on 6)

A host cell comprising the vector of claim 6.

### Claim 8 (depends on 6)

A plant tissue comprising the vector of claim 6.

### Claim 9 (depends on 8)

The plant tissue of claim 8 wherein said tissue is selected from the group consisting of leaf tissue, veins, petioles, small branches, branches, flowers, trunk, fruits and seeds.

### Claim 10

A small inhibitory ribonucleic acid molecule (siRNA) that inhibits expression of an L. invasa or O. maskelli nucleic acid molecule encoding coatamer subunit alpha (alpha COP), chromodomain helicase DNA binding protein, venom carboxylesterase 6 isoform 1, chitin synthase, ferritin or juvenile hormone epoxide hydrolase.

### Claim 11

A double stranded ribonucleic acid molecule (dsRNA) comprising a unit of a first strand of nucleotides that is substantially identical to at least 17 contiguous nucleotides set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26, SEQ ID NO: 45-123, SEQ ID NO: 127-132, SEQ ID NO: 234-244 and a second strand of nucleotides that is substantially complementary to said first strand of nucleotides.

### Claim 12 (depends on 11)

The dsRNA of claim 11 wherein said first strand of nucleotides is substantially identical to at least 17 contiguous nucleotides set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, or SEQ ID NO: 150-222.

### Claim 13 (depends on 11)

The dsRNA of claim 11 wherein said first and second strands of nucleotides are at least about 25, 35, 50, 70 or 100 nucleotides in length.

### Claim 14 (depends on 11)

The dsRNA according to claim 11 wherein over their respective lengths said first and second strands of nucleotides are 70-100% identical to SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 45-123, SEQ ID NO: 127-132 or SEQ ID NO: 234-244.

### Claim 15 (depends on 11)

The dsRNA according to claim 11 wherein the sequences of said first and second strands of nucleotides are less than about 80% identical to the sequence of the honey bee ortholog of said first and second strands of nucleotides.

### Claim 16 (depends on 11)

The dsRNA according to claim 11 comprising at least two (2) of said units.

### Claim 17 (depends on 16)

The dsRNA of claim 16 wherein said at least two units are derived from different sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 45-123, SEQ ID NO: 127-132, SEQ ID NO: 150-222 and SEQ ID NO: 234-244.

### Claim 18 (depends on 11)

The dsRNA according to claim 11 further comprising a loop region separating said first strand and said second strand nucleotides.

### Claim 19 (depends on 11)

A vector comprising an expression control sequence operatively linked to a nucleotide sequence that is a template for one or both strands the ds RNA according to claim 11.

### Claim 20 (depends on 19)

A host cell comprising the expression vector of claim 19.

### Claim 21 (depends on 20)

The host cell of claim 20 wherein said host is a bacterial cell or a yeast cell.

### Claim 22 (depends on 21)

The host cell of claim 21 wherein said host is an Agrobacterium.

### Claim 23 (depends on 22)

A plant tissue transformed with the host cell of claim 22.

### Claim 24 (depends on 11)

A plant tissue comprising the dsRNA of claim 11.

### Claim 25

A small inhibitory ribonucleic acid molecule (dsRNA) that inhibits expression of an essential gene of L. invasa or O. maskelli.

### Claim 26 (depends on 25)

The dsRNA of claim 25 comprising a unit of a first strand of nucleotides that is substantially identical to at least 17 contiguous nucleotides from said essential gene, and a second strand nucleotides that is substantially complementary to said first strand of nucleotides.

### Claim 27 (depends on 26)

The dsRNA of claim 26 wherein said first and second strands of nucleotides are at least about 25, 35, 50, 70 or 100 nucleotides in length.

### Claim 28 (depends on 25)

The dsRNA according to claim 25 wherein over their respective lengths said first and second strands of nucleotides are 70-100% identical to said essential gene.

### Claim 29 (depends on 26)

The dsRNA according to claim 26 comprising at least two (2) of said units.

### Claim 30 (depends on 29)

The dsRNA of claim 29 wherein said at least two units are derived from different essential genes.

### Claim 31 (depends on 30)

The dsRNA of claim 30 wherein said at least two units are derived from a single species.

### Claim 32 (depends on 30)

The dsRNA of claim 30 wherein said at least two units are derived from different species.

### Claim 33 (depends on 26)

The dsRNA according to claim 26 further comprising a loop region separating said first strand and said second strand nucleotides.

### Claim 34 (depends on 26)

A vector comprising an expression control sequence operatively linked to a nucleotide sequence that is a template for one or both strands of the ds RNA according to claim 26.

### Claim 35 (depends on 34)

A host cell comprising the expression vector of claim 34.

### Claim 36 (depends on 26)

A plant tissue comprising the dsRNA according to claim 26.

### Claim 37 (depends on 34)

A plant tissue comprising the vector of claim 34.

### Claim 38 (depends on 35)

A plant tissue comprising the host cell of claim 35.

### Claim 39 (depends on 11)

A method of producing a pest resistant plant comprising expressing the dsRNA according to claim 11 in said plant or propagating the dsRNA according to claim 11 in reproductive material of said plant.

### Claim 40 (depends on 39)

The method of claim 39 wherein said plant is Eucalyptus.

### Claim 41 (depends on 40)

The method of claim 40 wherein said pest is a gall wasp.

### Claim 42 (depends on 41)

The method of claim 41 wherein said pest is L. invasa or O. maskelli.

### Claim 43 (depends on 11)

A method of inhibiting a pest infestation comprising cultivating a plant comprising a dsRNA according to claim 11, to inhibit said infestation.

### Claim 44 (depends on 43)

The method of claim 43 wherein said plant is Eucalyptus.

### Claim 45 (depends on 44)

The method of claim 44 wherein said pest is L. invasa or O. maskelli.

### Claim 46 (depends on 11)

A method of producing a plant resistant to a plant pathogenic pest comprising:
(a) transforming a plant cell with a recombinant DNA construct or combination of constructs that express the dsRNA according to claim 11;
(b) regenerating a plant from the transformed plant cell; and
(c) growing the transformed plant cell under conditions suitable for the transcription said recombinant DNA construct,
said grown transformed plant thus being resistant to said pest compared to an untransformed plant.

### Claim 47 (depends on 46)

The method of claim 46 further comprising transforming said plant cell with a recombinant DNA construct that expresses a single stranded RNA that is complementary to one strand said dsRNA or a fragment thereof.

### Claim 48 (depends on 46)

The method of claim 46 wherein said plant is Eucalyptus.

### Claim 49 (depends on 48)

The method of claim 48 wherein said pest is L. invasa or O. maskelli.