# US 2014/0208455 A1 · Chimeric promoters capable of mediating gene expression in plants upon pathogen infection and uses thereof

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- Number: US 2014/0208455 A1
- Document ID: US20140208455A1
- Status: Published application
- Kind: A1
- Application number: 13/930,266
- Filed: 2013-06-28
- Published: 2014-07-24
- Earliest claimed: 1998-11-12
- Assignees: Max-Planck-Gesellschaft zur Foerderung der Wissenschaften e.V.
- Applicants: Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V.
- Inventors: Christoph Kirsch; Elke Logemann; Klaus Hahlbrock; Paul Rushton; Imre Somssich
- Web page: https://patentgrep.com/patent/US20140208455A1

## CPC

- C12N 15/8216: {Methods for controlling, regulating or enhancing expression of transgenes in plant cells}
- C07K 14/415: from plants
- C07K 16/16: against material from plants
- C12N 15/8239: {pathogen inducible}
- C12N 15/8263: {Ablation; Apoptosis}
- C12N 15/8279: {for biotic stress resistance, pathogen resistance, disease resistance}
- C12N 15/8283: {for virus resistance}
- C12Q 1/025: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics (antimicrobial activity C12Q1/18)}
- 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)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

Described are synthetic promoters capable of mediating gene expression in plants upon pathogen infection. Furthermore, recombinant genes and vectors comprising said chimeric promoters as well as host cells transformed with such chimeric promoters, recombinant genes, or vectors are provided. Additionally, diagnostic compositions and kits comprising such chimeric promoters, recombinant genes, vectors or cells are described. Provided are further methods for the identification of compounds being capable of activating or inhibiting genes that are specifically expressed in plants upon pathogen infection employing the above described means. Furthermore, transgenic plant cells, plant tissue, and plants containing the above-described chimeric promoters, recombinant genes, and vectors as well as the use of the aforementioned chimeric promoters, recombinant genes, vectors and/or compounds identified by the method of the invention in plant cell and tissue culture, plant breeding, and/or agriculture are described.

## Claims

### Claim 1

A chimeric promoter capable of local gene expression in plants of an operably linked nucleic acid sequence, wherein the expression is induced by a pathogen elicitor treatment, a pathogen infection, or both, wherein the chimeric promoter comprises:
(i) at least one cis-acting element sufficient to direct: pathogen-elicitor-specific expression of a nucleic acid sequence, pathogen-infection-specific expression of a nucleic acid, or both, wherein the cis-acting element comprises the nucleotide sequence of SEQ ID NO: 7 or 14, and
(ii) a minimal promoter.

### Claim 2 (depends on 1)

The chimeric promoter according to claim 1, further comprising at least one cis-acting element consisting of the nucleotide sequence selected from the group consisting of SEQ ID NO: 1-16.

### Claim 3 (depends on 1)

The chimeric promoter according to claim 1, wherein the cis-acting element comprises two copies of the nucleotide sequence of SEQ ID NO:7 or 14.

### Claim 4 (depends on 3)

The chimeric promoter according to claim 3, further comprising two copies of SEQ ID NO: 3 or 4.

### Claim 5 (depends on 2)

The chimeric promoter of claim 2, wherein said chimeric plant promoter comprises homo- and/or hetero-multimeric forms of said cis-acting element(s).

### Claim 6 (depends on 5)

The chimeric promoter of claim 5, wherein said multimeric form is a dimer or tetramer.

### Claim 7 (depends on 1)

The chimeric promoter of claim 1, wherein the minimal promoter is derived from the CaMV35S promoter, CHS promoter, PR1 promoter, or hcbt2 promoter.

### Claim 8 (depends on 1)

The chimeric promoter of claim 1, wherein the distance between said cis-acting element and said minimal promoter is 12 to 300 base pairs, more preferably 25 to 70 base pairs, and most preferably 40 to 60 base pairs.

### Claim 9 (depends on 2)

The chimeric promoter of claim 2 wherein at least two of said cis-acting elements are separated by a spacer, wherein the spacer is (a) from 4 to 10 or (b) about 50 to about 1000 base pairs.

### Claim 10 (depends on 1)

The chimeric promoter of claim 1, wherein the induction of gene expression upon elicitor treatment or pathogen infection is at least 10-fold, preferably at least 15-fold.

### Claim 11 (depends on 1)

A recombinant gene comprising the chimeric promoter of claim 1.

### Claim 12 (depends on 11)

The recombinant gene of claim 11, wherein the chimeric promoter is operatively linked to a heterologous DNA sequence.

### Claim 13 (depends on 11)

The recombinant gene of claim 11, wherein at least one of said cis-acting elements is located in the 5′- or 3′-untranslated region or in an intron of the recombinant gene.

### Claim 14 (depends on 12)

The recombinant gene of claim 12, wherein said heterologous DNA sequence encodes a (poly)peptide, cytotoxic protein, antibody, antisense RNA, sense RNA, ribozyme, transcription factor, protease, nuclease, lipase, or polymerase.

### Claim 15 (depends on 1)

A vector comprising the chimeric promoter of claim 1 or the recombinant gene thereof.

### Claim 16 (depends on 1)

A method for the production of a transgenic plant, a transgenic plant cell or transgenic plant tissue comprising introducing the chimeric promoter of claim 1, the recombinant gene thereof, or a vector derived therefrom into the genome of a plant, plant cell or plant tissue to produce the transgenic plant, the transgenic plant cell or the transgenic plant tissue.

### Claim 17 (depends on 1)

A transgenic plant cell comprising the chimeric promoter of claim 1, the recombinant gene thereof, or a vector derived therefrom.

### Claim 18 (depends on 17)

A transgenic plant or plant tissue comprising transgenic plant cells of claim 17.

### Claim 19 (depends on 18)

The transgenic plant of claim 18, which due to the presence of the chimeric promoter or the recombinant gene attained resistance or improved resistance against a pathogen the corresponding wild-type plant was susceptible to.

### Claim 20 (depends on 17, 18)

Harvestable parts of a transgenic plant of claim 18 comprising transgenic plant cells of claim 17.

### Claim 21 (depends on 17, 18)

Propagation material of a transgenic plant of claim 18 comprising transgenic plant cells of claim 17.

### Claim 22

An isolated cis-acting element sufficient to direct pathogen-elicitor-specific expression, pathogen-infection-specific expression, or both, consisting of the nucleotide sequence of SEQ ID NO: 7 or 14.

### Claim 23 (depends on 1)

A method for the identification of an activator or inhibitor of genes specifically expressed in plants upon pathogen infection comprising the steps of:
(a) providing a plant, plant cell, or plant tissue comprising a recombinant DNA molecule comprising a readout system operatively linked to the chimeric promoter of claim 1;
(b) culturing said plant cell or tissue or maintaining said plant in the presence of a compound or a sample comprising a plurality of compounds under conditions which permit expression of said readout system;
(c) identifying or verifying a sample and compound, respectively, which leads to suppression or activation and/or enhancement of expression of said readout system in said plant, plant cell, or plant tissue.

### Claim 24 (depends on 23)

The method of claim 23 further comprising the step of
(d) subdividing the samples identified in step (c) and repeating steps (a) to (c) one or more times.

### Claim 25 (depends on 23)

The method of claim 23 further comprising the step of
(e) identifying and/or isolating from the identified sample the compound responsible for said suppression or activation and/or enhancement of expression of said readout system in said plant, plant cell, or plant tissue.

### Claim 26 (depends on 1, 23)

The method of claim 23, wherein
(a) said recombinant DNA molecule is recombinant gene comprising the chimeric promoter of claim 1 or comprising the chimeric promoter of claim 1 or the recombinant gene thereof;
(b) said plant cell is a transgenic plant cell comprising the chimeric promoter of claim 1, the recombinant gene thereof, or a vector derived therefrom;
(c) said plant tissue is a plant tissue of comprising said transgenic plant cells, or
(d) said plant is a plant comprising said transgenic plant cells.

### Claim 27 (depends on 23)

A method for preparing a plant elicitor comprising the steps of the method of claim 23 and formulating the compound obtained or identified in step (c) or (e) in a form suitable for the application in agriculture or plant cell and tissue culture.

### Claim 28 (depends on 23)

A compound obtained or identified by the method of claim 23 which is an activator or inhibitor of gene expression and/or function in plants.

### Claim 29 (depends on 22, 28)

An antibody specifically recognizing the compound of claim 28 or the cis-acting element of claim 22.

### Claim 30 (depends on 28)

A plant protection composition comprising the compound of claim 28 and a botanically inert medium.

### Claim 31

A method of rendering a gene responsive to pathogens comprising inserting at least one cis-acting element sufficient to direct pathogen elicitor-specific expression, pathogen-infection-specific expression, or both into the promoter of said gene, wherein the at least one cis-acting element comprises the nucleotide sequence of SEQ ID NO: 7 or 14.

### Claim 32

A method for preparing a promoter capable of mediating local gene expression in plants upon pathogen infection comprising operably linking a cis-acting element sufficient to direct elicitor-specific expression to a transcription initiation sequence of a promoter, wherein the cis-acting element comprises the nucleotide sequence of SEQ ID NO: 7 or 14.

### Claim 33 (depends on 31)

The method of claim 31, further comprising inserting at least one cis-acting element consisting of the nucleotide sequence selected from the group consisting of SEQ ID NO: 1-16 or homo- and/or hetero-multimeric forms of said cis-acting element(s).

### Claim 34 (depends on 33)

The method of claim 33, further comprising deleting non-specific cis-acting elements in the promoter.

### Claim 35

A promoter obtained by a method of rendering a gene responsive to pathogens comprising inserting at least one cis-acting element sufficient to direct pathogen-elicitor-specific expression, pathogen-infection-specific expression, or both, into the promoter of said gene, wherein
(1) induction of local gene expression in plants upon elicitor treatment, pathogen infection, or both, is between 10-fold and 15-fold and wherein the at least one cis-acting element comprises SEQ ID NO:7 or 14, or
(2) induction of local gene expression in plants is at least 15 fold and the at least one cis-acting element comprises two copies of SEQ ID NO: 7 or 14, or a combination of one copy of SEQ ID NO:7 or 14 and one copy selected from the group consisting of SEQ ID NO: 1-16.

### Claim 36

An isolated chimeric promoter to render a gene responsive to pathogens, obtained by a method comprising
inserting at least one cis-acting element sufficient to direct pathogen-elicitor-induced expression, pathogen-infection induced expression, or both, of an operably linked nucleic acid, into the promoter of said gene, wherein the at least one cis-acting element comprises the nucleotide sequence of SEQ ID NO: 7 or 14.

### Claim 37 (depends on 36)

A recombinant gene wherein the isolated chimeric promoter of claim 36 is linked to a coding sequence for a gene.

### Claim 38 (depends on 36)

A vector wherein the isolated chimeric promoter of claim 36 is linked to vector sequences.

### Claim 39 (depends on 36)

A method for the production of a transgenic plant, a transgenic plant cell or a transgenic plant tissue, wherein the method comprises:
introducing the isolated chimeric promoter of claim 36 into the genome of a plant, plant cell or plant tissue to produce the transgenic plant, the transgenic plant cell or the transgenic plant tissue.