# US 2014/0208461 A1 · Defensin-encoding nucleic acid molecules derived from nicotiana alata, uses therefor and transgenic plants comprising same

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- Number: US 2014/0208461 A1
- Document ID: US20140208461A1
- Status: Published application
- Kind: A1
- Application number: 14/223,913
- Filed: 2014-03-24
- Published: 2014-07-24
- Earliest claimed: 2001-02-08
- Assignees: Hexima Limited
- Applicants: Hexima Limited
- Inventors: Marilyn Anne ANDERSON; Fung Tso LAY; Robyn Louise HEATH
- Web page: https://patentgrep.com/patent/US20140208461A1

## CPC

- C12N 15/8279: {for biotic stress resistance, pathogen resistance, disease resistance}
- A01N 37/46: N-acyl derivatives
- A01N 65/20: Fabaceae or Leguminosae [Pea or Legume family], e.g. pea, lentil, soybean, clover, acacia, honey locust, derris or millettia
- A01N 65/38: Solanaceae [Potato family], e.g. nightshade, tomato, tobacco or chilli pepper
- C07K 14/415: from plants
- C12N 15/8281: {for bacterial resistance}
- C12N 15/8282: {for fungal resistance}
- C12N 15/8283: {for virus resistance}
- C12N 15/8286: {for insect resistance}
- Y02A 40/146: Genetically Modified [GMO] plants, e.g. transgenic plants
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A01N: PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF (preservation of food or foodstuff A23); BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES (preparations for medical, dental or toiletry purposes which kill or prevent the growth or proliferation of unwanted organisms A61K); PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- 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 provides nucleic acid molecules derived from Nicotiana alata, which encode defensin-like molecules. The present invention contemplates the use of such nucleic acid molecules in the generation of transgenic plants having resistance or at least reduced sensitivity to plant pests including insects, microorganisms, fungi and/or viruses and the of the encoded defensin-like molecules in compositions for topical application to a plant or a plant part so as to reduce prevent or reduce infestation of the plant or plant part by plant pests. The transgenic plants provided by the present invention include monocotyledonous and dicotyledonous plants, and particularly include crop plants and ornamental flowering plants.

## Claims

### Claim 1

A method for generating a plant seed with increased or enhanced resistance to a plant pest, said method comprising introducing into the genome of a cell of a plant seed a genetic construct comprising a promoter or functional equivalent thereof operably linked to a nucleotide sequence encoding a floral-derived, defensin-like molecule having a mature domain comprising the amino acid sequence:—
a1a2CIa3a4a5a6a7a8a9a10a11a12CIIa13a14a15a16a17CIIIa18a19a20CIVa21a22a23a24a25a26a27a8
a29CVa30a31a32a33a34a35CVIa36CVIIa37a38a39CVIII
wherein “a” may be the same or different and represents any amino acid residue, the numerical subscript on each “a” represents its position in the amino acid sequence and “C” represents a cysteine residue at a position indicated by its Roman numeral and wherein said mature domain exhibits inhibitory activity against plant pests such as insect pests and regenerating a plant from said cell or group of cells.

### Claim 2 (depends on 1)

The method of claim 1 wherein the defensin-like molecule comprises a mature domain having the amino acid sequence set forth in SEQ ID NO:58.

### Claim 3 (depends on 2)

The method of claim 2 wherein the defensin-like molecule comprises a mature domain having an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:8 or an amino acid sequence having at least about 70% similarity to one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

### Claim 4 (depends on 3)

The method of claim 3 wherein the defensin-like molecule comprises the amino acid sequence set forth in SEQ ID NO:8.

### Claim 5 (depends on 1)

The method of claim 1 wherein the plant pest is an insect or a fungus.

### Claim 6

A transfected or transformed cell of a plant seed, said cell comprising a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a polypeptide comprising, in its precursor form, an N-terminal signal domain, a mature domain and an acidic C-terminal domain wherein said polypeptide is produced during flower development and its mature domain has activity against one or more plant pests.

### Claim 7 (depends on 6)

The transfected or transformed cell of claim 6 wherein the polypeptide comprises a mature domain having the structure:
a1a2CIa3a4a5a6a7a8a9a10a11a12CIIa13a14a15a16a17CIIIa18a19a20CIVa21a22a23a24a25a26a27a8
a29CVa30a31a32a33a34a35CVIa36CVIIa37a38a39CVIII
wherein “a” may be the same or different and represents any amino acid residue, the numerical subscript on each “a” represents its position in the amino acid sequence and “C” represents a cysteine residue at a position indicated by its Roman numeral and wherein the mature domain has activity against one or more plant pests.

### Claim 8 (depends on 7)

The transfected or transformed cell of claim 7 wherein the polypeptide comprises an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:8 or an amino acid sequence having at least about 70% similarity to one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

### Claim 9 (depends on 7)

The method of claim 7 wherein the defensin-like molecule comprises the amino acid sequence set forth in SEQ ID NO:8.

### Claim 10 (depends on 8)

Seeds of plants engineered to express the nucleic acid molecule encoding the defensin-like molecule of claim 8.

### Claim 11

A method for generating a plant seed with increased or enhanced resistance to a plant pathogenic fungus or insect, said method comprising introducing into the genome of a cell from a plant seed or genome of a group of cells from a plant seed a nucleic acid molecule encoding a defensin comprising an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:16 and SEQ ID NO:18, or an amino acid sequence set forth in any one of SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:16 and SEQ ID NO:18, together with a nucleic acid molecule encoding a proteinase inhibitor.

### Claim 12 (depends on 11)

The method of claim 11 wherein the nucleic acid molecule encoding the defensin comprises the nucleotide sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:12, SEQ ID NO:15 and SEQ ID NO:17.

### Claim 13 (depends on 11)

The method of claim 11 wherein the proteinase inhibitor comprises the amino acid sequence set forth in SEQ ID NO:57.

### Claim 14 (depends on 13)

The method of claim 13, wherein the nucleic acid encoding the proteinase inhibitor is set forth in SEQ ID NO:56.

### Claim 15 (depends on 11)

The method of claim 11, wherein the defensin comprises the amino acid sequence set forth in SEQ ID NO:8, and wherein the proteinase inhibitor comprises the amino acid sequence set forth in SEQ ID NO:57.

### Claim 16 (depends on 11)

The method of claim 11, wherein the amino acid sequence comprises SEQ ID NO:14, SEQ ID NO:16 or SEQ ID NO:18.

### Claim 17 (depends on 11)

The method of claim 11, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:13, SEQ ID NO:15 or SEQ ID NO:17.

### Claim 18

A transfected or transformed cell from a seed from a plant, said cell comprising a nucleic acid molecule encoding a defensin, defensin-like molecule or part thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or an amino acid sequence set forth in any one of SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:16 and SEQ ID NO:18, together with a nucleic acid molecule encoding a proteinase inhibitor.

### Claim 19 (depends on 18)

The transfected or transformed cell of claim 18, wherein the nucleic acid molecule encoding the defensin comprises the nucleotide sequence of SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:15 or SEQ ID NO:17.

### Claim 20 (depends on 18)

The transfected or transformed cell of claim 18, wherein the defensin comprises an amino acid sequence set forth in SEQ ID NO:14, SEQ ID NO:16 or SEQ ID NO:18, and wherein the proteinase inhibitor comprises the amino acid sequence set forth in SEQ ID NO:57.

### Claim 21 (depends on 18)

The transfected cell of claim 18, wherein the proteinase inhibitor has the amino acid sequence set forth in SEQ ID NO:57.

### Claim 22 (depends on 21)

The transfected or transformed cell of claim 21, wherein the defensin amino acid sequence is set forth in SEQ ID NO:18.

### Claim 23 (depends on 16)

A genetically modified plant seed, plant, plant plant partor plant progeny having reduced sensitivity to a plant pathogenic fungus or insect, said plant seed comprising the transfected or transformed cell of claim 16, wherein the defensin and the proteinase inhibitor are heterologous to the untransformed plant and wherein the reduced sensitivity of said genetically modified plant seed, plant, plant part or plant progeny is measured relative to a corresponding plant seed or plant untransformed with said nucleic acid molecule encoding the defensin, defensin-like molecule or part thereof together with said nucleic acid molecule encoding the proteinase inhibitor.

### Claim at position 24

24-30. (canceled)

### Claim 31 (depends on 16)

The transfected or transformed cell of claim 16, wherein the defensin comprises an amino acid sequence set forth in SEQ ID NO:14, SEQ ID NO:16 or SEQ ID NO:18, and wherein the proteinase inhibitor comprises the amino acid sequence set forth in SEQ ID NO;57.

### Claim 32

A composition comprising a defensin or defensin-like molecule alone or in combination with another agent.

### Claim 33 (depends on 32)

The composition of claim 32 wherein the defensin comprises an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:16 and SEQ ID NO:18 and the composition optionally further comprises a proteinase inhibitor.

### Claim 34 (depends on 32)

The composition of claim 32 which comprises a proteinase inhibitor.

### Claim 35 (depends on 34)

The composition of claim 34 wherein the proteinase inhibitor comprises the amino acid sequence set for the in SEQ ID NO:57.

### Claim 36 (depends on 33)

A method for controlling fungal infestation of a seed comprising contacting a seed with the composition of claim 33.