# US 2013/0117880 A1 · Transgenic plants expressing a viral antifungal protein

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- Number: US 2013/0117880 A1
- Document ID: US20130117880A1
- Status: Published application
- Kind: A1
- Application number: 13/809,997
- Filed: 2011-07-20
- Published: 2013-05-09
- Earliest claimed: 2010-07-20
- Assignees: Donald Danforth Plant Science Center
- Inventors: Thomas James Smith; Dilip Shah
- Web page: https://patentgrep.com/patent/US20130117880A1

## CPC

- C12N 15/8282: {for fungal resistance}
- A61P 31/10: Antimycotics
- C12N 2720/00033: Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

Transgenic plants expressing the KP4 antifungal protein are provided which exhibit high levels of antifungal resistance. Such transgenic plants contain a recombinant DNA construct comprising a heterologous signal peptide sequence that is oper ably linked to a non-native nucleic acid sequence encoding a mature KP4 antifungal protein.

## Claims

### Claim 1

A transgenic plant comprising a recombinant nucleic acid construct, said recombinant nucleic acid construct comprising a promoter that is operably linked to a nucleic acid encoding a heterologous signal peptide that is operably linked to a non-native nucleic acid encoding a KP4 antifungal protein that is operably linked to a polyadenylation sequence, wherein said transgenic plant expresses said KP4 protein in the apoplast and provides for at least 50% inhibition of a plant pathogenic fungal infection relative to a control plant that lacks said recombinant nucleic acid construct.

### Claim 2 (depends on 1)

The transgenic plant of claim 1, wherein said transgenic plant provides for at least 75%, at least 85%, or at least 95% inhibition of a plant pathogenic fungal infection relative to a control plant that lacks said recombinant nucleic acid construct.

### Claim 3 (depends on 1)

The transgenic plant of claim 1, wherein said transgenic plant provides for at least 50% inhibition of a biotrophic plant pathogenic fungus and/or at least 50% inhibition of a necrotrophic plant pathogenic fungus.

### Claim 4 (depends on 3)

The transgenic plant of claim 3, wherein said biotrophic plant pathogenic fungus is selected from the group consisting of Ustilago species, Podosphaera species, Erysiphe species, Phakopsora species, and Puccinia species.

### Claim 5 (depends on 3)

The transgenic plant of claim 3, wherein said necrotrophic plant pathogenic fungus is selected from the group consisting of Alternaria species, Botrytis species, Colletotrichum species, Cercospora species, Fusarium species, Phoma species, Phytophthora species, Pythium species, Sclerotinia species, and Verticillium species.

### Claim 6 (depends on 3)

The transgenic plant of claim 3, wherein said transgenic plant is a monocot plant or a dicot plant and wherein said non-native nucleic acid sequence comprises one or more non-native codons that are more abundant in monocot plant genes and/or one or more non-native codons that are more abundant in dicot plant genes.

### Claim 7 (depends on 1)

The transgenic plant of claim 1, wherein said plant is a monocot plant is selected from the group consisting of barley, corn, flax, oat, rice, rye, sorghum, turf grass, sugarcane, and wheat.

### Claim 8 (depends on 1)

The transgenic plant of claim 1, wherein said plant is a dicot plant is selected from the group consisting of alfalfa, Arabidopsis, barrel medic, banana, broccoli, bean, cabbage, canola, carrot, cassava, cauliflower, celery, citrus, cotton, a cucurbit, eucalyptus, garlic, grape, onion, lettuce, pea, peanut, pepper, potato, poplar, pine, sunflower, safflower, soybean, strawberry, sugar beet, sweet potato, tobacco, and tomato.

### Claim 9 (depends on 1, 2, 3, 4, 5, 6, 7, 8)

The transgenic plant of any one of claims 1-8, wherein said plant further comprises a second recombinant nucleic acid construct that provides for expression of MsDef1, MtDef2, MtDef4, Rs-AFP1, or Rs-AFP2.

### Claim 10 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9)

The transgenic plant of any one of claims 1-9, wherein said heterologous signal peptide is a signal peptide of a plant gene.

### Claim 11 (depends on 10)

The transgenic plant of claim 10, wherein said plant gene is a dicot or a monocot plant gene.

### Claim 12 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11)

A transgenic plant cell obtained from the transgenic plant of any one of claims 1-11.

### Claim 13 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11)

A transgenic plant seed obtained from the transgenic plant of any one of claims 1-11.

### Claim 14 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13)

A processed food or feed composition obtained from either:
a) the transgenic plant seed of claim 13; or,
b) a transgenic plant part selected from the group consisting of a leaf, a stem, a flower, a root, and a tuber obtained from the transgenic plant of any one of claims 1-11.

### Claim 15 (depends on 14)

The processed food or feed composition of claim 14, wherein said food or feed composition is a meal, a flour, an oil, or a starch.

### Claim 16 (depends on 14)

The processed food or feed composition of claim 14, wherein mycotoxin levels in said food or feed composition are reduced by at least 50% relative to processed food or feed composition that lacks said recombinant nucleic acid construct.

### Claim 17 (depends on 16)

The processed food or feed composition of claim 16, wherein mycotoxin levels in said food or feed composition are reduced by at least 75%, at least 85%, or at least 95% relative to processed food or feed composition that lacks said recombinant nucleic acid construct.

### Claim 18 (depends on 16, 17)

The processed food or feed composition of claim 16 or 17, wherein said mycotoxin is an aflatoxin, a fumonisin, a vomitoxin, or a trichothecene.

### Claim 19 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11)

A method of producing the transgenic plant of any one of claims 1-11, comprising the steps of:
i) introducing a recombinant nucleic acid construct comprising a promoter that is operably linked to a nucleic acid encoding a heterologous signal peptide that is operably linked to a non-native nucleic acid encoding a KP4 antifungal protein that is operably linked to a polyadenylation sequence into a plant, a plant cell, or a plant tissue; and
ii) selecting for a transgenic plant comprising said recombinant nucleic acid construct, wherein said transgenic plant selected in step (b) plant expresses said KP4 antifungal protein in the apoplast and provides for at least 50% inhibition of a plant pathogenic fungal infection relative to a control plant that lacks said recombinant nucleic acid construct.

### Claim 20 (depends on 19)

The method of claim 19, wherein said transgenic plant is a monocot plant or a dicot plant.

### Claim 21 (depends on 19)

The method of claim 19, wherein said nucleic acid construct is introduced into said plant, a plant cell or a plant tissue in step (a) by a method selected from the group consisting of particle bombardment, DNA transfection, DNA electroporation, Agrobacterium-mediated, Rhizobium-mediated, and Sinorhizobium-mediated transformation.

### Claim 22 (depends on 19)

The method of claim 19, wherein said nucleic acid construct further comprises a sequence encoding a selectable marker and wherein said transgenic plant is obtained in step (b) by growing said plant, plant cell, or plant tissue under conditions requiring expression of said selectable marker for plant growth.

### Claim 23 (depends on 19)

The method of claim 19, wherein said plant pathogenic fungus is selected from the group consisting of an Alternaria sp., an Ascochyta sp., a Botrytis sp.; a Cercospora sp., a Colletotrichum sp., a Diplodia sp., an Erysiphe sp., a Fusarium sp., Gaeumanomyces sp., Helminthosporium sp., Magnaporthe grisea, Macrophomina sp., a Nectria sp., a Peronospora sp., Phakopsora pachyrhizi, Phialophora gregata, a Phoma sp., a Phymatotrichum sp., a Phytophthora sp., a Plasrnopara sp., a Puccinia sp., a Podosphaera sp., a Pyrenophora sp., a Pyricularia sp, a Pythium sp., a Rhizoctonia sp., a Scerotium sp., a Sclerotinia sp., a Septoria sp., Stenocarpella maydis, a Thielaviopsis sp., an Uncinula sp, a Venturia sp., and a Verticillium sp.

### Claim 24 (depends on 13)

A method for obtaining the transgenic seed of claim 13, comprising the steps of:
a) crossing a transgenic plant comprising a recombinant nucleic acid construct, said recombinant nucleic acid construct comprising a promoter that is operably linked to a nucleic acid encoding a heterologous signal peptide that is operably linked to a non-native nucleic acid encoding a KP4 antifungal protein that is operably linked to a polyadenylation sequence, wherein said transgenic plant expresses said KP4 protein in the apoplast and provides for at least 50% inhibition of a plant pathogenic fungal infection relative to a control plant that lacks said recombinant nucleic acid construct to a plant that lacks said recombinant nucleic acid construct; and,
b) harvesting seed from a pollen recipient from said cross of step (i), thereby obtaining said transgenic seed.

### Claim 25 (depends on 24)

The method of claim 24, wherein said method further comprises the steps of
iii) selecting for said transgenic seed from said harvested seed; and/or
iv) screening for said transgenic seed from said harvested seed.

### Claim 26 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11)

A method of inhibiting a plant pathogenic fungal infection of a transgenic plant comprising the steps of:
i. exposing a transgenic plant of any one of claims 1-11 to a plant pathogenic fungus; and,
ii. obtaining a transgenic plant that exhibits at least 50% inhibition of a plant pathogenic fungal infection relative to a control plant that lacks said recombinant nucleic acid construct.

### Claim 27 (depends on 26)

The method of claim 26, wherein said plant pathogenic fungus is selected from the group consisting of an Alternaria sp., an Ascochyta sp., a Botrytis sp.; a Cercospora sp., a Colletotrichum sp., a Diplodia sp., an Erysiphe sp., a Fusarium sp., Gaeumanomyces sp., Helminthosporium sp., Magnaporthe grisea, Macrophomina sp., a Nectria sp., a Peronospora sp., Phakopsora pachyrhizi, Phialophora gregata, a Phoma sp., a Phymatotrichum sp., a Phytophthora sp., a Plasrnopara sp., a Puccinia sp., a Podosphaera sp., a Pyrenophora sp., a Pyricularia sp, a Pythium sp., a Rhizoctonia sp., a Scerotium sp., a Sclerotinia sp., a Septoria sp., Stenocarpella maydis, a Thielaviopsis sp., an Uncinula sp, a Venturia sp., and a Verticillium sp.

### Claim 28 (depends on 26, 27)

The method of claim 26 or 27, wherein said transgenic plant provides for at least 75%, at least 85%. or at least 95% inhibition of a plant pathogenic fungal infection relative to a control plant that lacks said recombinant nucleic acid construct.

### Claim 29 (depends on 26)

The method of claim 26, wherein said method further comprises the step of harvesting at least one plant part selected from the group consisting of a leaf, a stem, a flower, a root, a tuber, or a seed from said plant obtained in step (ii).

### Claim 30 (depends on 29)

The method of claim 29, wherein mycotoxin levels in said plant part are reduced by at least 50%, at least 75%, at least 85%, or at least 95% relative to a plant part obtained from a control plant that lacks said recombinant nucleic acid construct.

### Claim 31 (depends on 29)

The method of claim 29, wherein said when said method further comprises the step of obtaining a processed food or feed composition from said plant part.

### Claim 32 (depends on 31)

The method of claim 31, wherein mycotoxin levels in said processed food or feed composition from said plant part are reduced by at least 50%, at least 75%, at least 85%, or at least 95% relative to a processed food or feed composition obtained from a plant part of a control plant that lacks said recombinant nucleic acid construct.

### Claim 33

A transgenic maize plant comprising a chromosome containing an insertion of a KP4 protein expression cassette of plasmid pZP212-KP4, wherein said insertion provides for at least 50% inhibition of a plant pathogenic fungal infection relative to a control maize plant that lacks said recombinant nucleic acid construct.

### Claim 34 (depends on 33)

The transgenic maize plant of claim 33, wherein said transgenic maize plant is selected from the group consisting of transgenic maize plant lines 851, 947, 1040, 885, and 8510.

### Claim 35

A transgenic soybean plant comprising a chromosome containing an insertion of an FMV promoter to a chimeric KP4 protein encoding region of SEQ ID NO:7, wherein said insertion provides for at least 50% inhibition of a plant pathogenic fungal infection relative to a control soybean plant that lacks said recombinant nucleic acid construct.

### Claim 36 (depends on 35)

The transgenic soybean plant of claim 35, wherein said transgenic soybean plant is selected from the group consisting of transgenic soybean plant lines 2, 3, 7, 9, 10, 14, and 16.