# US 2017/0253884 A1 · Use of micropeptides in order to stimulate mycorrhizal symbiosis

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- Number: US 2017/0253884 A1
- Document ID: US20170253884A1
- Status: Published application
- Kind: A1
- Application number: 15/315,472
- Filed: 2015-06-03
- Published: 2017-09-07
- Earliest claimed: 2014-06-03
- Assignees: Universite Toulouse III - Paul Sabatier; Centre National De La Recherche Scientifique
- Applicants: Universite Toulouse III-Paul Sabatier; Centre National De La Recherche Scientifique
- Inventors: Jean-Philippe COMBIER; Dominique LAURES-SERGUES; Guillaume BECARD
- Web page: https://patentgrep.com/patent/US20170253884A1

## CPC

- C12N 15/8216: {Methods for controlling, regulating or enhancing expression of transgenes in plant cells}
- A01H 3/04: by treatment with chemicals
- A01N 37/46: N-acyl derivatives
- C07K 14/415: from plants
- C12N 1/14: Fungi (culture of mushrooms A01G18/00; as new plants A01H15/00); Culture media therefor
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 15/8218: {Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]}
- C12N 15/8241: {Phenotypically and genetically modified plants via recombinant DNA technology}
- C12N 15/8261: {with agronomic (input) traits, e.g. crop yield}
- C12N 15/8271: {for stress resistance, e.g. heavy metal resistance}
- C12N 15/8273: {for drought, cold, salt resistance}
- C12N 2310/141: MicroRNAs, miRNAs
- 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)
- A01H: NEW PLANTS OR {NON-TRANSGENIC} PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- 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

A method for promoting mycorrhizal symbiosis between a plant and a fungus includes using micropeptides (peptides encoded by microRNAs or “miPEPs”).

## Claims

### Claim at position 1

1-23. (canceled)

### Claim 24

A method for promoting mycorrhizal symbiosis, in particular arbuscular mycorrhizal symbiosis, between a plant and a fungus, comprising a step of introducing exogenously a miPEP into the plant,
said miPEP also being naturally present in said plant,
said miPEP introduced exogenously being a peptide comprising, or consisting of, a sequence identical to that of said miPEP naturally present,
said miPEP naturally present is a peptide of 3 to 100 amino acids the sequence of which is encoded by an open reading frame situated at 5′ on the primary transcript of a miRNA,
said miPEP being capable of modulating the accumulation of said miRNA in said plant, which miRNA regulates the expression of at least one gene involved in the arbuscular mycorrhizal symbiosis in said plant,
the sum of the quantity of said miPEP introduced exogenously and that of said miPEP naturally present being strictly greater than the quantity of said miPEP naturally present,
in particular, said gene involved in the mycorrhizal symbiosis encoding a transcription factor of the GRAS family,
in particular, said gene involved in the mycorrhizal symbiosis being selected from the group consisting of: HAM1 and HAM2.

### Claim 25 (depends on 24)

The method according to claim 24, in which said miRNA is:
miR171b, in particular in which said miR171b has a nucleotide sequence consisting of SEQ ID NO: 1,
slmiR171e, in particular in which said slmiR171e has a nucleotide sequence consisting of SEQ ID NO: 5,
ljmiR171b, in particular in which said ljmiR171b has a nucleotide sequence consisting of SEQ ID NO: 9, or
osmiR171i, in particular in which said osmiR171i has a nucleotide sequence consisting of SEQ ID NO: 13.

### Claim 26 (depends on 24)

The method according to claim 24, in which said miPEP is:
miPEP171b, in particular, in which said miPEP171b has an amino acid sequence consisting of SEQ ID NO: 2,
slmiPEP171e, in particular in which said slmiPEP171e has an amino acid sequence consisting of SEQ ID NO: 6,
ljmiPEP171b, in particular in which said ljmiPEP171b has an amino acid sequence consisting of SEQ ID NO: 10, or
osmiPEP171i, in particular in which said osmiPEP171i has an amino acid sequence consisting of SEQ ID NO: 14.

### Claim 27 (depends on 24)

The method according to claim 24, in which said plant is a monocotyledon plant, a dicotyledon plant, a solanaceous plant or a leguminous plant, in particular selected from: Medicago truncatula, Medicago sativa (alfalfa), Solanum lycopersicum (tomato), Lotus japonicus (birdsfoot trefoil) or Oryza sativa (rice).

### Claim 28 (depends on 24)

The method according to claim 24, in which said fungus is a glomeromycete or basidiomycete or ascomycete, preferably a glomeromycete.

### Claim 29 (depends on 24)

The method according to claim 24, for promoting the arbuscular mycorrhizal symbiosis between a Medicago truncatula plant and a glomeromycete fungus, in which the miPEP171b is introduced exogenously into said M. truncatula plant, said miPEP171b also being naturally present in said M. truncatula plant,
said miPEP171b introduced exogenously being a peptide the sequence of which comprises or consists of a sequence identical to that of said miPEP naturally present 171b, said sequence of the miPEP171b naturally present being encoded by an open reading frame situated at 5′ on the primary transcript of the miR171b, said miR171b regulating the expression of at least one gene involved in mycorrhizal symbiosis in M. truncatula,
the sum of the quantity of said miPEP171b introduced exogenously and that of said miPEP171b naturally present being strictly greater than the quantity of said miPEP171b naturally present in said Medicago truncatula plant.

### Claim 30 (depends on 24)

The method according to claim 24, for promoting the arbuscular mycorrhizal symbiosis between a Solanum lycopersicum plant and a glomeromycete fungus, in which slmiPEP171e is introduced exogenously into said Solanum lycopersicum plant, said slmiPEP171e also being naturally present in said Solanum lycopersicum plant,
said slmiPEP171e introduced exogenously being a peptide the sequence of which comprises or consists of a sequence identical to that of said slmiPEP171e naturally present, said sequence of the slmiPEP171e naturally present being encoded by an open reading frame situated at 5′ on the primary transcript of the slmiR171e, said slmiR171e regulating the expression of at least one gene involved in the mycorrhizal symbiosis in Solanum lycopersicum,
the sum of the quantity of said slmiPEP171e introduced exogenously and that of said slmiPEP171e naturally present being strictly greater than the quantity of said slmiPEP171e naturally present in said Solanum lycopersicum plant.

### Claim 31 (depends on 24)

The method according to claim 24, for promoting the arbuscular mycorrhizal symbiosis between a Lotus japonicus plant and a glomeromycete fungus, in which the ljmiPEP171b is introduced exogenously into said Lotus japonicus plant, said ljmiPEP171b also being naturally present in said Lotus japonicus plant,
said ljmiPEP171b introduced exogenously being a peptide the sequence of which comprises or consists of a sequence identical to that of said ljmiPEP171b naturally present, said sequence of the ljmiPEP171b naturally present being encoded by an open reading frame situated at 5′ on the primary transcript of the ljmiR171b, said ljmiR171b regulating the expression of at least one gene involved in mycorrhizal symbiosis in Lotus japonicus,
the sum of the quantity of said ljmiPEP171b introduced exogenously and that of said ljmiPEP171b naturally present being strictly greater than the quantity of said ljmiPEP171b naturally present in said Lotus japonicas plant.

### Claim 32 (depends on 24)

The method according to claim 24, for promoting the arbuscular mycorrhizal symbiosis between an Oryza sativa plant and a glomeromycete fungus, in which osmiPEP171i is introduced exogenously into said Oryza sativa plant, said osmiPEP171i also being naturally present in said Oryza sativa plant, said osmiPEP171i introduced exogenously being a peptide the sequence of which comprises or consists of a sequence identical to that of said naturally present osmiPEP171i, said sequence of the osmiPEP171i naturally present being encoded by an open reading frame situated at 5′ on the primary transcript of the osmiR171i, said miR171i regulating the expression of at least one gene involved in the mycorrhizal symbiosis in Oryza sativa, the sum of the quantity of said osmiPEP171i introduced exogenously and that of said osmiPEP171i naturally present being strictly greater than the quantity of said osmiPEP171i naturally present in said Oryza sativa plant.

### Claim 33 (depends on 24)

The method according to claim 24, in which said miRNA is:
miR171b, said miR171b having in particular a nucleotide sequence consisting of SEQ ID NO: 1,
said miPEP being in particular miPEP171b, said miPEP171b having in particular an amino acid sequence consisting of SEQ ID NO: 2,
slmiR171e, said slmiR171e having in particular a nucleotide sequence consisting of SEQ ID NO: 5,
said miPEP being in particular slmiPEP171e, said slmiPEP171e having in particular an amino acid sequence consisting of SEQ ID NO: 6,
ljmiR171b, said ljmiR171b having in particular a nucleotide sequence consisting of SEQ ID NO: 9,
said miPEP being in particular ljmiPEP171b, said ljmiPEP171b having in particular an amino acid sequence consisting of SEQ ID NO: 10, or
osmiR171i, said osmiR171i having in particular a nucleotide sequence consisting of SEQ ID NO: 13,
said miPEP being in particular osmiPEP171i, said osmiPEP171i having in particular an amino acid sequence consisting of SEQ ID NO: 14,
said fungus being in particular a glomeromycete,
said plant being in particular a monocotyledon plant, a dicotyledon plant, a solanaceous plant or a leguminous plant.

### Claim 34 (depends on 24)

The method according to claim 24, in which said miPEP is introduced into the plant:
externally, preferably by watering, by spraying or by adding a fertilizer, said miPEP being in particular administered to the plant in the form of a composition comprising 10−9 M to 10−4 M of said miPEP, in particular 10−9, 10−8, 10−7, 10−6, 10−5 or 10−4 M of said miPEP,
externally, preferably by watering, by spraying or by adding a fertilizer, said miPEP being in particular administered to a grain or a seed in the form of a composition comprising 10−9 M to 10−4 M of said miPEP, in particular 10−9, 10−8, 10−7, 10−6, 10−5 or 10−4 M of said miPEP, or
by means of a nucleic acid encoding said miPEP, said nucleic acid being introduced into the plant.

### Claim 35

A method for the production of a transgenic plant comprising:
a) a step of introducing a nucleic acid encoding for a miPEP of 3 to 100 amino acids into a plant, or into at least one cell of said plant, under conditions allowing the expression of said miPEP,
said miPEP also being naturally present in said plant, said miPEP naturally present is a peptide the sequence of which is encoded by an open reading frame situated at 5′ on the primary transcript of a miRNA, said miPEP being capable of modulating the accumulation of said miRNA in the plant, which miRNA regulates the expression of at least one gene involved in mycorrhizal symbiosis, in particular arbuscular mycorrhizal symbiosis, and
b) a step of growing the plant, or at least one cell of said plant, obtained in step a) under conditions allowing a transgenic plant to be obtained,
said gene involved in mycorrhizal symbiosis being in particular selected from the group consisting of: HAM1 and HAM2,
said miRNA being in particular miR171b, said miR171b having in particular a nucleotide sequence consisting of SEQ ID NO: 1,
said miPEP being in particular miPEP171b, said miPEP171b having in particular an amino acid sequence consisting of SEQ ID NO: 2,
said miRNA being in particular slmiR171e, said slmiR171e having in particular a nucleotide sequence consisting of SEQ ID NO: 5,
said miPEP being in particular slmiPEP171e, said slmiPEP171e having in particular an amino acid sequence consisting of SEQ ID NO: 6,
said miRNA being in particular ljmiR171b, said ljmiR171b having in particular a nucleotide sequence consisting of SEQ ID NO: 9,
said miPEP being in particular ljmiPEP171b, said ljmiPEP171b having in particular an amino acid sequence consisting of SEQ ID NO: 10, or
said miRNA being in particular osmiR171i, said osmiR171i having in particular a nucleotide sequence consisting of SEQ ID NO: 13,
said miPEP being in particular osmiPEP171i, said osmiPEP171i having in particular an amino acid sequence consisting of SEQ ID NO: 14.

### Claim 36 (depends on 35)

A transgenic plant as obtained by the methods as defined according to claim 35.

### Claim 37

A composition comprising:
miPEP171b as the active ingredient, said miPEP171b preferably consisting of SEQ ID NO: 2, said miPEP171b being in particular at a concentration of 10−9 M to 10−4 M, in particular 10−9, 10−8, 10−7, 10−6, 10−5 or 10−4 M,
slmiPEP171e as the active ingredient, said slmiPEP171e preferably consisting of SEQ ID NO: 6, said slmiPEP171e being in particular at a concentration of 10−9 M to 10−4 M, in particular 10−9, 10−8, 10−7, 10−6, 10−5 or 10−4 M,
ljmiPEP171b as the active ingredient, said ljmiPEP171b preferably consisting of SEQ ID NO: 10, said ljmiPEP171b being in particular at a concentration of 10−9 M to 10−4 M, in particular 10−9, 10−8, 10−7, 10−6, 10−5 or 10−4 M, or
osmiPEP171i as the active ingredient, said osmiPEP171i preferably consisting of SEQ ID NO: 14, said osmiPEP171i being in particular at a concentration of 109M to 10−4M, in particular 10−9, 10−8, 10−7, 10−6, 10−5 or 10−4 M,
in particular, said composition also comprising an excipient, a diluent or a solvent,
in particular, said composition being formulated so as to form a coating.

### Claim 38

A composition comprising in combination a quantity of seeds of a plant and a quantity of a peptide the sequence of which comprises or consists of a sequence identical to that of a miPEP naturally present in said plant,
said peptide having in particular a sequence comprising or consisting of a sequence identical to that of miPEP171b, of slmiPEP171e, of LjmiPEP171b or of osmiPEP171i,
in particular, said composition being so as to form a coated seed.

### Claim 39

A method for the culture of mycorrhizal fungi, in particular arbuscular mycorrhizal fungi, comprising a step of placing said fungi in contact with:
a mixture comprising a plant or a part of a plant, in particular a root culture, and a peptide the sequence of which comprises or consists of a sequence identical to that of a miPEP naturally present in said plant, said miPEP naturally present is a peptide the sequence of which is encoded by an open reading frame situated at 5′ on the primary transcript of a miRNA, said miPEP being capable of modulating the accumulation of said miRNA, which miRNA regulates the expression of at least one gene involved in mycorrhizal symbiosis in said plant, or
with a transgenic plant as defined previously,
the plant, the part of a plant, and the transgenic plant being suitable for forming mycorrhizal symbiosis with said fungus.

### Claim 40

A method for producing an inoculum of mycorrhizal fungi, in particular an inoculum of arbuscular mycorrhizal fungi, comprising:
a step of co-culture of fungi with a living plant material, called plant host, at least partially corresponding to a constitutive root part of a plant suitable for forming a symbiosis with said fungi, and
a step of placing a quantity of a peptide in contact with the aforementioned co-culture,
said peptide having a sequence comprising or consisting of a sequence identical to that of a miPEP naturally present in said plant, said miPEP naturally present is a peptide the sequence of which is encoded by an open reading frame situated at 5′ on the primary transcript of a miRNA, said miPEP being capable of modulating the accumulation of said miRNA, which miRNA regulates the expression of at least one gene involved in the mycorrhizal symbiosis in said plant.

### Claim 41

An inoculum of mycorrhizal fungi, in particular an inoculum of arbuscular mycorrhizal fungi, suitable for the inoculation of a plant host, comprising at least: a fungus and a peptide the sequence of which comprises or consists of a sequence identical to that of a miPEP naturally present in the plant host,
said miPEP naturally present in the plant host being a peptide the sequence of which is encoded by an open reading frame situated at 5′ on the primary transcript of a miRNA, said miPEP being capable of modulating the accumulation of said miRNA, which miRNA regulates the expression of at least one gene involved in mycorrhizal symbiosis in said plant host.