# US 11,299,745 B1 · Herbicide resistance genes

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- Number: US 11,299,745 B1
- Document ID: US11299745B1
- Status: Granted
- Kind: B1
- Application number: 17/403,476
- Filed: 2021-08-16
- Published: 2022-03-31
- Granted: 2022-04-12
- Earliest claimed: 2004-04-30
- Assignees: Dow AgroSciences LLC
- Applicants: Dow Agrosciences LLC
- Inventors: Terry Wright; Justin Lira; Donald Merlo; Nicole Arnold
- Web page: https://patentgrep.com/patent/US11299745B1

## CPC

- C12N 15/8274: {for herbicide resistance}
- C12N 9/0069: {acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)}
- C12N 9/0071: {acting on paired donors with incorporation of molecular oxygen (1.14)}
- C12N 15/8209: {Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The subject invention provides novel plants that are not only resistant to 2,4-D and other phenoxy auxin herbicides, but also to aryloxyphenoxypropionate herbicides. Heretofore, there was no expectation or suggestion that a plant with both of these advantageous properties could be produced by the introduction of a single gene. The subject invention also includes plants that produce one or more enzymes of the subject invention alone or “stacked” together with another herbicide resistance gene, preferably a glyphosate resistance gene, so as to provide broader and more robust weed control, increased treatment flexibility, and improved herbicide resistance management options. More specifically, preferred enzymes and genes for use according to the subject invention are referred to herein as AAD (aryloxyalkanoate dioxygenase) genes and proteins. No α-ketoglutarate-dependent dioxygenase enzyme has previously been reported to have the ability to degrade herbicides of different chemical classes and modes of action. This highly novel discovery is the basis of significant herbicide tolerant crop trait opportunities as well as development of selectable marker technology. The subject invention also includes related methods of controlling weeds. The subject invention enables novel combinations of herbicides to be used in new ways. Furthermore, the subject invention provides novel methods of preventing the formation of, and controlling, weeds that are resistant (or naturally more tolerant) to one or more herbicides such as glyphosate.

## Claims

### Claim 1

A transgenic soybean plant cell comprising a recombinant polynucleotide that encodes an AAD-1 protein that exhibits aryloxyalkanoate dioxygenase activity wherein said activity enzymatically degrades a phenoxy auxin herbicide and an (R)-aryloxyphenoxypropionate herbicide, which are aryloxyalkanoate herbicides, further wherein said AAD-1 protein comprises:
i) an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 9; and
ii) an AAD-1 motif having the general formula of:
HX112D(X)114-137T(X)139-269H(X)271-280R (SEQ ID NO: 34), wherein
X112 represents a single amino acid at position 112, relative to the sequence of SEQ ID NO: 9;
(X)114-137 represents a sequence of 24 amino acids;
(X)139-269 represents a sequence of 131 amino acids; and
(X)271-280 represents a sequence of 10 amino acids,
wherein said AAD-1 protein when expressed in a soybean plant cell renders said soybean plant cell tolerant to a phenoxy auxin herbicide and an (R)-aryloxyphenoxypropionate herbicide, as compared to an untransformed soybean plant cell.

### Claim 2 (depends on 1)

The plant cell of claim 1 wherein said AAD-1 motif has the general formula of: HX112D(X)114-137T(X)139-269H(X)271-280R(X)282-284R (SEQ ID NO: 35), wherein (X)282-284 represents a sequence of 3 amino acids.

### Claim 3 (depends on 1)

A transgenic soybean plant comprising a plurality of the soybean plant cell of claim 1, wherein expression of said polynucleotide renders said plant tolerant to said aryloxyalkanoate herbicide.

### Claim 4 (depends on 3)

The plant of claim 3 wherein said aryloxyalkanoate herbicide selected from the group consisting of 2,4-dichlorophenoxyacetic acid, MCPA, dichlorprop, and mecoprop.

### Claim 5 (depends on 3)

The plant of claim 3 wherein said aryloxyalkanoate herbicide is an (R)-aryloxyphenoxypropionate.

### Claim 6 (depends on 3)

The plant of claim 3 wherein said aryloxyalkanoate herbicide is selected from the group consisting of (R)-fluazifop, (R)-haloxyfop, (R)-diclofop, (R)-quizalofop, (R)-fenoxaprop, (R)-metamifop, (R)-cyhalofop, and (R)-clodinofop.

### Claim 7 (depends on 3)

The plant of claim 3 wherein said plant further comprises a second herbicide resistance gene.

### Claim 8 (depends on 7)

The plant of claim 7 wherein said second herbicide resistance gene renders said plant resistant to an herbicide selected from the group consisting of glyphosate, glufosinate, acetolactate synthase (ALS) inhibitors, inhibitors of 4-hydroxyphenyl-pyruvate-dioxygenase (HPPD), dicamba, and inhibitors of protoporphyrinogen oxidase (PPO).

### Claim 9 (depends on 3)

A method of controlling at least one weed in a field, wherein said field contains at least one plant of claim 3 wherein said method comprises applying to at least a portion of said field a first herbicide selected from the group consisting of a phenoxy auxin herbicide and an (R)-aryloxyphenoxypropionate herbicide.

### Claim 10 (depends on 9)

The method of claim 9 wherein said phenoxy auxin herbicide is an R-enantiomer of a chiral phenoxy auxin.

### Claim 11 (depends on 9)

The method of claim 9 wherein said phenoxy auxin herbicide is an achiral phenoxy auxin selected from the group consisting of 2,4-D and MCPA.

### Claim 12 (depends on 9)

The method of claim 9 wherein said (R)-aryloxyphenoxypropionate herbicide is selected from the group consisting of (R)-fluazifop, (R)-haloxyfop, (R)-diclofop, (R)-quizalofop, (R)-fenoxaprop, (R)-metamifop, (R)-cyhalofop, and (R)-clodinofop.

### Claim 13 (depends on 9)

The method of claim 9 wherein said method comprises applying a second herbicide.

### Claim 14 (depends on 13)

The method of claim 13 wherein said first herbicide and said second herbicide are applied sequentially.

### Claim 15 (depends on 13)

The method of claim 13 wherein said first herbicide and said second herbicide are applied concurrently.

### Claim 16 (depends on 13)

The method of claim 13 wherein said first herbicide is a phenoxy auxin and said second herbicide is an (R)-aryloxyphenoxypropionate.

### Claim 17 (depends on 13)

The method of claim 13 wherein said second herbicide is selected from the group consisting of glyphosate, glufosinate, dicamba, acetolactate synthase (ALS) inhibitors, protoporphyrinogen oxidase (PPO) inhibitors, and 4-hydroxyphenyl-pyruvate-dioxygenase (HPPD) inhibitors.

### Claim 18 (depends on 13)

The method of claim 13, wherein said first herbicide is 2,4-D and said second herbicide is glyphosate or glufosinate.

### Claim 19 (depends on 13)

The method of claim 13, wherein said first herbicide is an (R)-aryloxyphenoxypropionate and said second herbicide is glyphosate or glufosinate.

### Claim 20 (depends on 13)

The method of claim 13 wherein said plant further comprises a second herbicide resistance gene that renders said plant resistant to said second herbicide.

### Claim 21 (depends on 20)

The method of claim 20 wherein said second herbicide resistance gene is selected from the group consisting of a modified acetolactate synthase (ALS) gene, a glyphosate resistance gene, a glufosinate resistance gene, and a dicamba-degrading enzyme gene.

### Claim 22 (depends on 13)

The method of claim 13 wherein said method further comprises applying a third herbicide.

### Claim 23 (depends on 22)

The method of claim 22, wherein said third herbicide is selected from the group consisting of glyphosate, glufosinate, HPPD-inhibitors, PPO-inhibitors, ALS inhibitors, and dicamba.

### Claim 24 (depends on 23)

The method of claim 23 wherein said first, second and third herbicides are 2,4-D, quizalofop, and glyphosate.

### Claim 25 (depends on 1)

A seed comprising a plurality of the plant cell of claim 1.

### Claim 26 (depends on 25)

A method of controlling weeds in a field, wherein said method comprises applying an aryloxyalkanoate herbicide to said field and planting a seed of claim 25 in said field within 14 days after applying said aryloxyalkanoate herbicide.

### Claim 27 (depends on 25)

A plant grown from the seed of claim 25, wherein said plant comprises said polynucleotide.

### Claim 28 (depends on 27)

A part, progeny, or asexual propagate of the plant of claim 27, wherein said part, progeny, or sexual propagate comprises said polynucleotide.

### Claim 29

An (R)-aryloxyphenoxypropionate herbicide tolerant transgenic soybean plant cell comprising a recombinant polynucleotide that encodes an AAD-1 protein that exhibits aryloxyalkanoate dioxygenase activity wherein said activity enzymatically degrades a phenoxy auxin herbicide and an (R)-aryloxyphenoxypropionate herbicide, further wherein said AAD-1 protein comprises:
i) an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 9; and
ii) an AAD-1 motif having the general formula of:
HX112D(X)114-137T(X)139-269H(X)271-280R (SEQ ID NO: 34), wherein
X112 represents a single amino acid at position 112, relative to the sequence of SEQ ID NO: 9;
(X)114-137 represents a sequence of 24 amino acids;
(X)139-269 represents a sequence of 131 amino acids; and
(X)271-280 represents a sequence of 10 amino acids, wherein said motif has 90% sequence identity with corresponding amino acids of position 111 to 281 of SEQ ID NO: 9
wherein said AAD-1 protein when expressed in a soybean plant cell renders said soybean plant cell tolerant to a phenoxy auxin herbicide and an (R)-aryloxyphenoxypropionate herbicide, as compared to an untransformed soybean plant cell.