NOVEL METHOD FOR PROMOTING NODULATION IN PLANTS

Description

[0001] The present invention relates to the use of micropeptides (microRNA-encoded peptides or "miPEPs") to promote nodulation in plants.

[0002] In plants, the acquisition of nutrients from the soil is of major importance and can constitute a limitation to their development. Some plants have thus developed the capacity to establish symbiotic associations with soil microorganisms in order to improve nutrient acquisition.

[0003] Leguminous plants, for example, are capable of establishing a symbiosis with bacteria of the family Rhizobiaceae.The host plant and the bacterium will thus recognize each other through an exchange of molecular signals. In particular, the plant will produce flavonoids that will induce the expression of specific genes in the bacterium, leading to the production of lipochito-oligosaccharide compounds, Nod factors, which are at the origin of numerous developmental changes in the host plant. The bacteria will then penetrate the hairs of the plant roots, and induce the formation of nodules. This mechanism of colonization of the plant by the bacterium is called "nodulation."

[0004] The plant then provides the bacteria with both nutrients and an energy source in the form of adenosine triphosphate (ATP) generated by photosynthesis. In return, the bacteria fix atmospheric nitrogen in the form of ammonium, providing an important source of nitrogen for the plant to assimilate.

[0005] Nitrogen is a nutrient whose supply is often crucial for plant growth. Generally speaking, the higher the nitrogen fixation in plants, the higher the yield. Consequently, the higher the nodulation rate in plants is often associated with a higher yield. Efficient plant nodulation also reduces fertilizer requirements and provides soils with nitrogen compounds that can be used by plants. Furthermore, the protein content of leguminous plants is also higher than in other plant families, due to nitrogen fixation by bacteria. This high protein content makes leguminous plants particularly important for both human consumption and animal feed.

[0006] Inoculating plants with specific strains of bacteria can promote nodulation. However, although various techniques are available, inoculation is often ineffective. Therefore, it is necessary to provide a simple treatment to improve plant nodulation.

[0007] MicroRNAs (miRs) are small non-coding RNAs, approximately 21 nucleotides in length after maturation, that control the expression of target genes at the post-transcriptional level, by degrading the target mRNA or inhibiting its translation. MiRs are particularly common in plants, and the genes targeted by miRs are often key genes in developmental processes.

[0008] The regulation of miR expression is very little known, but it has been shown that it involves, like most coding genes, an RNA polymerase II: this enzyme produces a primary transcript, called “pri-miR”,which is then matured by a protein complex containing Dicer-type enzymes. This maturation first leads to the formation of a miR precursor called “pre-miR”, having a stem-loop secondary structure containing the miR and its sequence miR* complementary. Then the precursor is matured, leading to the formation of a shorter double-stranded RNA containing the miR and the miR*. The miR is then taken up by the RISC complex which cleaves the mRNA of the target gene or inhibits its translation.

[0009] In particular, miR172c has been identified in leguminous plants such as alfalfa, trefoil, soybean, and bean (Lelandais-Brière et al., Genome-wide Medicago truncatula small RNA analysis revealed novel microRNAs and isoforms differentially regulated in roots and nodules, Plant Cell 21: 2780-2796, 2009; Wang et al., Identification and expression analysis of miRNAs from nitrogen-fixing soybean nodules, Biochem Biophys Res Commun 378: 799-803, 2009; Valdes-Lôpez et al., Essential role of MYB transcription factor: PvPHR1 and microRNA: PvmiR399 in phosphorus deficiency signaling in common bean roots, Plant Cell Environ 31: 1834-1843, 2008; De Luis et al., Two microRNAs linked to nodule infection and nitrogen-fixing ability in the vegetable Lotus japonicus, Plant Physiol 160: 2137-2154, 2012).

[0010] Recently, miR172c has been shown to regulate nodule formation by reducing the expression of its target gene. NNC1(Nodule Number Control 1), a gene transcription factor AP2, which reduces gene expression NOD40, essential in the nodulation process in soybean (Wang et al., Soybean miR172c targets the repressive AP2 transcription factor NNC1 to activate ENOD40 expression and regulate module initiation, Plant Cell 26: 4782-4801, 2014). Another recent study also showed that an increase in the amount of miR172c leads to an increase in nodulation and nitrogen fixation in beans (Nova-Franco et al., The miR172c-AP2-1 Node as a Key Regulator of the Common Bean - Rhizobia Nitrogen Fixation Symbiosis, Plant Physiol. doi:10.1104 / pp.114.255547, March 2015).

[0011] Until now, miRs, and by extension their primary transcript, have always been considered, due to their particular mode of action, as non-coding regulatory RNAs that do not produce any peptide. However, the inventors have recently demonstrated in patent application FR 13 / 60727 the existence of micropeptides (or "miPEPs", microRNA encoded PEPtides) capable of modulating the accumulation of miRs in cells.

[0012] In this context, the present invention aims to propose new effective and ecological tools to promote nodulation in plants.

[0013] One aspect of the invention is to propose a new use of miPEPs to promote nodulation between a plant and a bacterium.

[0014] Another aspect of the invention relates to a miPEP for promoting plant nodulation.

[0015] Another aspect of the invention also relates to a novel method of growing plants in symbiosis with a bacterium.

[0016] Another aspect of the invention is to provide a composition of miPEPs making it possible to promote nodulation between a plant and a bacterium.

[0017] The invention also relates to a transgenic plant and parts of transgenic plants, their production method, as well as organs, cells and seeds of transgenic plants, ecologically modified plants, and bacterial inocula.

[0018] In summary of the following, the invention is as defined in claims 1 to 15.

[0019] The invention therefore relates to the use of a peptide for promoting nodulation between a plant and a bacterium, said peptide being introduced into the plant, said peptide having an amino acid sequence comprising or consisting of a sequence identical to that of a miPEP naturally present in said plant, said miPEP naturally present in said plant being a peptide of 3 to 100 acids, in particular of 4 to 100 amino acids, the sequence of which is encoded by an open reading frame located on the primary transcript of a miR, said miPEP being capable of modulating the accumulation of said miR in said plant, which miR regulates the expression of at least one gene involved in nodulation in said plant.

[0020] Surprisingly and unexpectedly, the Inventors found that the use of peptides whose sequence comprises or consists of a sequence identical to that of miPEPs encoded on the primary transcripts of miRs, makes it possible to promote nodulation between a plant and a bacterium.

[0021] In the invention, the terms “microRNA”, “non-coding microRNA” And “miR” are equivalent and can be used interchangeably. They define small RNA molecules of about 21 nucleotides, which are not translated and do not lead to a peptide or protein. However, in this mature form, miRs perform a regulatory function for certain genes way post-transcriptional mechanisms, such as through the RISC complex.

[0022] THE “miR primary transcript” (Or « pri-miR") corresponds to the RNA molecule directly obtained from the transcription of the DNA molecule. Generally, this primary transcript undergoes one or more post-transcriptional modifications, which result, for example, in a particular structure of the RNA or cleavage of certain parts of the RNA by splicing phenomena, and which lead to the precursor form of the miR or (" pre-miR ”), then to the mature form of miR

[0023] The terms “micropeptides” And “miPEPs” ( microRNA encoded PEPtides ) are equivalent and can be used interchangeably. They define a peptide that is encoded by an open reading frame present on the primary transcript of a miR, and which is capable of modulating the accumulation of said miR. MiPEPs within the meaning of the present invention should not be understood as necessarily being small peptides, since “micro” does not correspond to the size of the peptide.

[0024] As indicated in patent application FR 13 / 60727, the content of which must be considered as part of the present application, miPEPs are peptides: from 4 to 100 amino acids, preferably from 4 to 60 amino acids, in particular from 4 to 59 amino acids, encoded by an open reading frame contained in the primary transcript of a miR, preferably in the 5' part of the primary transcript of said miR, and capable of modulating the accumulation of said miR in a eukaryotic cell.

[0025] The terms " open reading frame » Or " ORF » (open reading frame) are equivalent and can be used interchangeably. They correspond to a sequence of nucleotides in a DNA or RNA molecule that can potentially code for a peptide or protein: the said open reading frame begins with a start codon (the start codon generally coding for a methionine), followed by a series of codons (each codon coding for an amino acid), and ends with a stop codon (the stop codon not being translated).

[0026] In the invention, the ORFs may be specifically named “miORFs” when these are present on primary miR transcripts.

[0027] The miORFs as defined in the invention may have a size of 15 to 303 nucleotides. Since an amino acid is encoded by a codon of 3 nucleotides, miORFs of 15 to 303 nucleotides encode miPEPS of 4 to 100 amino acids.

[0028] In particular, miORFs have a size of: 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 47, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 267, 270, 273, 276, 279, 282, 285, 288, 291, 294, 297, 300 or 303 nucleotides, and respectively encode miPEPs having a size from: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 amino acids.

[0029] A miPEP can also be 3 to 100 amino acids in size.

[0030] Given the degeneracy of the genetic code, the same miPEP can be encoded by several nucleotide sequences. Such nucleotide sequences, different from each other by at least one nucleotide but encoding the same peptide, are called “degenerate sequences”.

[0031] In the invention, the term « plant » refers generally to all or part of a plant at any stage of development (including the plant in seed or young shoot form), one or more plant organs (such as leaves, roots, stem, flowers), one or more plant cells, or a cluster of plant cells.

[0032] The expression “nodulation” refers to the colonization of a plant by a symbiont bacterium resulting in the formation of nodules on the roots.

[0033] In a non-limiting manner, the parameters allowing to determine and quantify the nodulation between a plant and a bacterium can be in particular: the size and number of nodules, the nitrogen content in all or part of the plant, or indirectly, the development of certain parts of the plant, such as pods or roots.

[0034] THE "nodules'' can also be called "nodosités". These are blisters that form on plant roots under the action of bacteria. Their size and number can be measured by eye or by microscope, using techniques known to those skilled in the art. Their size is measured after long-term cultivation (more than 30 days) by measuring the size of nodules on photographs, for example using software such as ImageJ.

[0035] There "nitrogen content"corresponds to the nitrogen concentration in the plant or in a part of the plant. For example, it may be the nitrogen concentration in the aerial parts in g of nitrogen per kg of dry weight of leaves. This nitrogen concentration can be determined by techniques known to those skilled in the art. For example, one method involves burning a sample of known mass at a high temperature (about 900°C) in the presence of oxygen, leading to the release of carbon dioxide, water and nitrogen. The gases are then passed over special columns (such as an aqueous solution of potassium hydroxide) which absorb the carbon dioxide and water. A column containing a thermal conductivity detector at the end is then used to separate the nitrogen from any residual carbon dioxide and water and the residual nitrogen content is quantified.

[0036] THE "development of certain parts of the plant"corresponds to the growth or maturation of certain parts of the plant. The development of certain parts of the plant can, for example, be determined by measuring the growth rate or the size or weight of certain parts of the plant. This parameter can be easily measured by, for example, measuring the dry weight of roots, pods or aerial parts.

[0037] Furthermore, in the invention, the expression “The favorite nodulation”, or “improve nodulation,” noted : either an acceleration of nodulation (such as for example a faster formation of nodules in a given plant compared to a reference plant), or an increase in nodulation (such as for example a greater number or a greater size of nodules in a given plant compared to a reference plant), or an acceleration and an increase in nodulation.

[0038] It is important to note that the use according to the invention has the advantage of being ecological, because miPEP is a peptide which is naturally present in the plant.

[0039] The invention also relates to the use of a miPEP introduced exogenously into a plant to promote nodulation between said plant and a bacterium, said exogenously introduced miPEP being a peptide comprising, or consisting of, a sequence identical to that of a miPEP naturally present in said plant, which naturally present miPEP is a peptide of 3 to 100 amino acids, in particular of 4 to 100 amino acids, the sequence of which is encoded by an open reading frame located 5' on the primary transcript of a miR, said miPEP being capable of modulating the accumulation of said miR in said plant, which miR regulates the expression of at least one gene involved in nodulation in said plant, the sum of the quantity of said exogenously introduced miPEP and that of said naturally present miPEP being strictly greater than the quantity of said naturally present miPEP.

[0040] In the invention, the expression " exogenously introduced miPEP » refers to a miPEP artificially introduced into the plant whether or not it exists naturally in the plant.

[0041] The introduction of a miPEP exogenously into the plant therefore involves a technical step, which step is not a natural phenomenon and does not correspond to either crossbreeding or selection.

[0042] The exogenously introduced miPEP may be either a peptide produced outside the plant (such as an isolated and / or purified peptide, a synthetic peptide or a recombinant peptide), or a peptide produced in the plant following the unnatural introduction of a nucleic acid encoding said miPEP into said plant.

[0043] If miPEP exists naturally in the plant, it is a " miPEP of endogenous origin”. If miPEP does not exist naturally in the plant, it is a "miPEP of exogenous origin".

[0044] When introduced into the plant a " miPEP of exogenous origin,” it is then necessary to also introduce the corresponding miR and its primary transcript.

[0045] The plant into which the miPEP has not been introduced has a basal amount of said miPEP, which corresponds to that of said naturally occurring miPEP. The use of a miPEP comprising, or consisting of, a sequence identical to that of said miPEP results in an increase in the total amount of miPEP, which modulates the accumulation of the miR whose primary transcript contains the sequence coding for said miPEP.

[0046] Furthermore, the introduced miPEP is found in the plant and its introduction has no impact on its stability.

[0047] In the invention, by " accumulation,” we mean the production of a molecule, such as a miR or a miPEP, in the cell.

[0048] So, the "modulation of accumulation" of a molecule in a cell corresponds to a modification of the quantity of this molecule in the cell.

[0049] Furthermore, the effect of a miPEP can be observed via the modulation of miR accumulation, but also through the modulation of the accumulation of the pri-miR or the corresponding pre-miR.

[0050] In one embodiment, the invention relates to the use as defined above, wherein the modulation of the accumulation of said miR is a decrease or an increase in the accumulation of said miR, in particular an increase.

[0051] A " decreased miR accumulation» corresponds to a decrease in the quantity of said molecule in the cell.

[0052] Conversely, a "increased miR accumulation" corresponds to an increase in the quantity of said molecule in the cell.

[0053] In one embodiment, the invention relates to the use as defined above in which said gene involved in nodulation encodes a transcription factor of the AP2 family.

[0054] In one embodiment, the invention relates to the use as defined above, wherein said gene involved in nodulation is the transcription factor NNC1.

[0055] In one embodiment, the invention relates to the use as defined above, wherein said gene involved in nodulation is the gene NSP1.

[0056] In one embodiment, the invention relates to the use as defined above, wherein said gene involved in nodulation is the gene NIN.

[0057] In one embodiment, the invention relates to the use as defined above, wherein said gene involved in nodulation is the gene ENOD40-1.

[0058] In one embodiment, the invention relates to the use as defined above, wherein said gene involved in nodulation is the gene Hb2.

[0059] In one embodiment, the invention relates to the use as defined above, wherein said gene involved in nodulation is the gene nifH.

[0060] In particular, the invention relates to the use as defined above, wherein said miR172c has a nucleotide sequence having at least 80% identity, preferably at least 90% identity, with the nucleotide sequence SEQ ID NO: 1.

[0061] In one embodiment, the invention relates to the use as defined above, wherein said miRNA is miR172c, in particular, wherein said miR172c has a nucleotide sequence comprising or consisting of SEQ ID NO: 1.

[0062] In particular, the invention relates to the use as defined above, wherein said miPEP172c has an amino acid sequence having at least 80% identity, preferably at least 90% identity, with the amino acid sequence SEQ ID NO: 2.

[0063] In one embodiment, the invention relates to the use as defined above, wherein said miPEP is miPEP172c, in particular, wherein said miPEP172c has an amino acid sequence comprising or consisting of SEQ ID NO: 2.

[0064] In particular, the use as defined above is described, wherein said miR167c has a nucleotide sequence having at least 80% identity, preferably at least 90% identity, with the nucleotide sequence SEQ ID NO: 6.

[0065] In one embodiment, the use as defined above is described, wherein said miRNA is miR167c, in particular, wherein said miR167c has a nucleotide sequence comprising or consisting of SEQ ID NO: 6.

[0066] In particular, the use as defined above is described, wherein said miPEP167c has an amino acid sequence having at least 80% identity, preferably at least 90% identity, with the amino acid sequence SEQ ID NO: 7.

[0067] In one embodiment, described is the use as defined above, wherein said miPEP is miPEP167c, in particular, wherein said miPEP167c has an amino acid sequence comprising or consisting of SEQ ID NO: 7.

[0068] In one embodiment, the invention relates to the use as defined above, wherein said plant is a leguminous plant, such as Lotus (Lotus sp.) soybeans (Glycine max), peanut (Arachis hypogaea), the bean (Phaseolus vulgaris), the pea (Pisum sativum), the lens ( Culinary lenses), chickpea (Cicer arietinum), broad bean and field bean ( Vicia faba ), vetches (Vicia sp.), vetch (Lathyrus sp.), alfalfa (Medicago sp.), clover (Clover sp.), lupin (Lupinus sp.), mung bean ( Radiated vine), licorice (Glycyrrhiza glabra), rosewood (Dalbergia), the birdsfoot trefoil (Lotus corniculatus), sainfoin (Onobrychis viciifolia ), rooibos (Aspalathus linearis), fenugreek (Trigonella fenugreek ).

[0069] In one embodiment, the invention relates to the use as defined above, wherein said plant is sugar beet ( Beetroot ).

[0070] In one embodiment, the invention relates to the use as defined above, wherein said plant is chosen from Medicago truncatula, M. sativa And Glycine max.

[0071] In one embodiment, the invention relates to the use as defined above, wherein said plant is Glycine max.

[0072] In one embodiment, the invention relates to the use as defined above, wherein said bacterium is a bacterium of the family of Rhizobiaceae.

[0073] In a particular embodiment, the invention relates to the use as defined above, in which said bacterium is chosen from the genera Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium Or Azorhizobium.

[0074] In the invention, said bacterium may also be chosen from: Rhizobium alamii, Rhizobium alkalisoli, Rhizobium cellulosilyticum, Rhizobium daejeonense, Rhizobium endophyticum, Rhizobium etli, Rhizobium galegae, Rhizobium gallicum, Rhizobium giardinii, Rhizobium hainanense, Rhizobium herbae, Rhizobium huautlense, Rhizobium indigoferae, Rhizobium leguminosarum, Rhizobium loessense, Rhizobium lusitanum, Rhizobium mesosinicum, Rhizobium miluonense, Rhizobium mongolense, Rhizobium multihospitium, Rhizobium oryzae, Rhizobium phaseoli, Rhizobium pisi, Rhizobium tibeticum, Rhizobium sullae, Rhizobium tropici, Rhizobium tubonense, Rhizobium undicola, Rhizobium vignae, Rhizobium yanglingense, Mesorhizobium albiziae, Mesorhizobium alhagi, Mesorhizobium amorphae, Mesorhizobium australicum, Mesorhizobium camelthorni, Mesorhizobium caraganae, Mesorhizobium chacoense, Mesorhizobium cicero, Mesorhizobium gobiense, Mesorhizobium huakuii, Mesorhizobium loti, Mesorhizobium mediterraneum, Mesorhizobium metallidurans, Mesorhizobium opportunistum, Mesorhizobium plurifarium, Mesorhizobium robiniae, Mesorhizobium shangrilense, Mesorhizobium septentrionale, Mesorhizobium tarimense, Mesorhizobium temperatum, Mesorhizobium tianshanense Ensifer abri, Sinorhizobium americanum, Ensifer arboris, Ensifer fredii, Ensifer garamanticus, Ensifer indiaense, Ensifer kostiensis, Ensifer kummerowiae, Ensifer medicae, Ensifer meliloti, Ensifer mexicanus, Sinorhizobium morelense, Ensifer adhaerens, Ensifer numidicus, Ensifer saheli, Ensifer sojae, Ensifer terangae, Bradyrhizobium canariense, Bradyrhizobium denitrificans, Bradyrhizobium elkanii, Bradyrhizobium iriomotense, Bradyrhizobium japonicum, Bradyrhizobium jicamae, Bradyrhizobium liaoningense, Bradyrhizobium pachyrhizi, Bradyrhizobium yuanmingense Burkholderia caribensis, Burkholderia cepacia, Burkholderia mimosarum, Burkholderia nodosa, Burkholderia phymatum, Burkholderia sage, Burkholderia tuberum Phyllobacterium trifolii, Phyllobacterium ifriqiyense, Phyllobacterium leguminum Microvirga lupine, Microvirga lotononidis, Microvirga zambiensis Azorhizobium caulinodans, Azorhizobium doebereinerae Ochrobactrum cytisi, Ochrobactrum lupini Methylobacterium nodulans, Cupriavidus taiwanensis, Devotions of Neptune, Shinella kummerowiae.

[0075] In one embodiment, the invention relates to the use as defined above, wherein said bacterium is a bacterium selected from Rhizobium leguminosarum, Rhizobium etli, Rhizobium tropici, Rhizobium galegae, Sinorhizobium sp. NGR234, Sinorhizobium meliloti, Sinorhizobium fredii, Sinorhizobium saheli, Sinorhizobium teranga, Mesorhizobium ciceri, Mesorhizobium huakuii, Mesorhizobium loti, Bradyrhizobium elkanii, Bradyrhizobium japonicum, Bradyrhizobium lupini, Bradyrhizobium sp. "cowpea" and Azorhizobium caulinodans.

[0076] In particular, the bacteria Rhizobium elti allows the nodulation of the bean.

[0077] In particular, the bacteria Rhizobium leguminosarum allows nodulation of peas, alfalfa, sugar beet, vetch, lentil.

[0078] In particular, the bacteria Rhizobium pisi allows nodulation of peas, beans, vetch.

[0079] In particular, the bacteria Rhizobium phaseoli allows the nodulation of dry beans, beans.

[0080] In particular, the bacteria Rhizobium fabae allows the nodulation of the bean.

[0081] In particular, the bacteria Rhizobium ciceri allows the nodulation of the chickpea.

[0082] In particular, the bacteria Rhizobium trifoli allows the nodulation of clover.

[0083] In particular, the bacteria Sinorhizobium meliloti allows the nodulation of alfalfa, fenugreek or sweet clover.

[0084] In particular, the bacteria Sinorhizobium fredii allows nodulation of soybeans.

[0085] In particular, the bacteria Mesorhizobium loti allows the nodulation of lotus, clover, lupin, or chickpea.

[0086] In particular, the bacteria Bradyrhizobium japonicum allows nodulation of soybeans.

[0087] In particular, the bacteria Bradyrhizobium sp. Vigna allows the nodulation of the peanut.

[0088] In particular, the bacteria Bradyrhizobium sp. Arachis allows the nodulation of the peanut.

[0089] In one embodiment, the invention relates to the use as defined above, for promoting nodulation between a plant Glycine max and a bacterium Bradirhizobium japonicum, wherein miPEP172c is introduced exogenously into said plant Glycine max, said miPEP172c also being naturally present in said plant Glycine max, said exogenously introduced miPEP172c being a peptide whose sequence comprises or consists of a sequence identical to that of said naturally occurring miPEP172c, said sequence of naturally occurring miPEP172c being encoded by an open reading frame located 5' on the primary transcript of miR172c, which miR172c regulates the expression of at least one gene involved in nodulation in Glycine max, the sum of the quantity of said miPEP172c introduced exogenously and that of said miPEP172c naturally present being strictly greater than the quantity of said miPEP172c naturally present in said plant Glycine max.

[0090] In one embodiment, the invention relates to the use of a miPEP to promote nodulation between a plant Glycine max and a bacterium Bradirhizobium japonicum, wherein miPEP172c is introduced exogenously into said plant Glycine max, said miPEP172c also being naturally present in said plant Glycine max, said exogenously introduced miPEP172c being a peptide whose sequence has at least 80% identity, preferably at least 90% identity with the sequence SEQ ID NO: 2, said sequence of the naturally occurring miPEP172c being encoded by an open reading frame located 5' on the primary transcript of miR172c, which miR172c regulates the expression of at least one gene involved in nodulation in Glycine max, the sum of the quantity of said miPEP172c introduced exogenously and that of said miPEP172c naturally present being strictly greater than the quantity of said miPEP172c naturally present in said plant Glycine max.

[0091] In one embodiment, the invention relates to the use of a miPEP to promote nodulation between a plant Glycine max and a bacterium Bradirhizobium japonicum, wherein miPEP172c is introduced exogenously into said plant Glycine max, said miPEP172c also being naturally present in said plant Glycine max, said exogenously introduced miPEP172c being a peptide whose sequence comprises or consists of SEQ ID NO: 2, said sequence of naturally occurring miPEP172c being encoded by an open reading frame located 5' on the primary transcript of miR172c, which miR172c regulates the expression of at least one gene involved in nodulation in Glycine max, the sum of the quantity of said miPEP172c introduced exogenously and that of said miPEP172c naturally present being strictly greater than the quantity of said miPEP172c naturally present in said plant Glycine max.

[0092] In one embodiment, the invention relates to the use of a miPEP to promote nodulation between a plant Glycine max and a bacterium Bradirhizobium japonicum, wherein miPEP172c is introduced exogenously into said plant Glycine max, said miPEP172c also being naturally present in said plant Glycine max, said exogenously introduced miPEP172c being a peptide whose sequence comprises or consists of a sequence identical to that of said naturally occurring miPEP172c, said sequence of naturally occurring miPEP172c being encoded by an open reading frame located 5' on the primary transcript of miR172c, which miR172c regulates the expression of at least one gene involved in nodulation in Glycine max, which miR172c comprises or consists of SEQ ID NO: 1, the sum of the quantity of said exogenously introduced miPEP172c and that of said naturally present miPEP172c being strictly greater than the quantity of said naturally present miPEP172c in said plant Glycine max.

[0093] In one embodiment, the use as defined above is described, for promoting nodulation between a plant Glycine max and a bacterium Bradirhizobium japonicum, wherein miPEP167c is introduced exogenously into said plant Glycine max, said miPEP167c also being naturally present in said plant Glycine max, said exogenously introduced miPEP167c being a peptide whose sequence comprises or consists of a sequence identical to that of said naturally occurring miPEP167c, said sequence of naturally occurring miPEP167c being encoded by an open reading frame located 5' on the primary transcript of miR167c, which miR167c regulates the expression of at least one gene involved in nodulation in Glycine max, the sum of the quantity of said miPEP167c introduced exogenously and that of said miPEP167c naturally present being strictly greater than the quantity of said miPEP167c naturally present in said plant Glycine max.

[0094] In one embodiment, the use of a miPEP to promote nodulation between a plant Glycine max and a bacterium Bradirhizobium japonicum, wherein miPEP167c is introduced exogenously into said plant Glycine max, said miPEP167c also being naturally present in said plant Glycine max, said exogenously introduced miPEP167c being a peptide whose sequence has at least 80% identity, preferably at least 90% identity with the sequence SEQ ID NO: 7, said sequence of the naturally occurring miPEP167c being encoded by an open reading frame located 5' on the primary transcript of miR167c, which miR167c regulates the expression of at least one gene involved in nodulation in Glycine max, the sum of the quantity of said miPEP167c introduced exogenously and that of said miPEP167c naturally present being strictly greater than the quantity of said miPEP167c naturally present in said plant Glycine max.

[0095] In one embodiment, the use of a miPEP to promote nodulation between a plant Glycine max and a bacterium Bradyrhizobium japonicum, wherein miPEP167c is introduced exogenously into said plant Glycine max,said miPEP167c also being naturally present in said plant Glycine max, said exogenously introduced miPEP167c being a peptide whose sequence comprises or consists of SEQ ID NO: 7, said sequence of the naturally occurring miPEP 167c being encoded by an open reading frame located 5' on the primary transcript of miR167c, which miR167c regulates the expression of at least one gene involved in nodulation in Glycine max, the sum of the quantity of said miPEP167c introduced exogenously and that of said miPEP167c naturally present being strictly greater than the quantity of said miPEP167c naturally present in said plant Glycine max.

[0096] In one embodiment, the use of a miPEP to promote nodulation between a plant Glycine max and a bacterium Bradyrhizobium japonicum, wherein miPEP167c is introduced exogenously into said plant Glycine max,said miPEP167c also being naturally present in said plant Glycine max, said exogenously introduced miPEP167c being a peptide whose sequence comprises or consists of a sequence identical to that of said naturally occurring miPEP167c, said sequence of naturally occurring miPEP167c being encoded by an open reading frame located 5' on the primary transcript of miR167c, which miR167c regulates the expression of at least one gene involved in nodulation in Glycine max, which miR167c comprises or consists of SEQ ID NO: 6, the sum of the quantity of said exogenously introduced miPEP167c and that of said naturally present miPEP167c being strictly greater than the quantity of said naturally present miPEP167c in said plant Glycine max.

[0097] In one embodiment, the invention relates to the use as defined above, wherein said miPEP is introduced externally into the plant, preferably by watering, spraying or by adding a fertilizer, potting soil, growing substrate or using a support in contact with the plant.

[0098] In one embodiment, the invention relates to the use as defined above, wherein said miPEP is introduced externally into a seed or a seed, preferably by watering, by spraying or by adding a fertilizer, a potting soil, a growing substrate or using a support in contact with the seed or the seed.

[0099] In one embodiment, the invention relates to the use as defined above, wherein said miPEP is used to treat the plant in seed form.

[0100] In one embodiment, the invention relates to the use as defined above, in which said miPEP is introduced by watering, in particular by spraying.

[0101] In one embodiment, the invention relates to the use as defined above, wherein said miPEP is introduced by watering and by the addition of a fertilizer.

[0102] In one embodiment, the invention relates to the use as defined above, wherein said miPEP is introduced by the addition of a fertilizer.

[0103] In one embodiment, the invention relates to the use as defined above, wherein said miPEP is introduced, by watering, and by adding a fertilizer.

[0104] In one embodiment, the invention relates to the use as defined above, wherein said miPEP is introduced by the addition of granules.

[0105] The inventors have indeed unexpectedly found that it is possible to directly apply a composition comprising a miPEP to the plant to modulate the accumulation of the corresponding miR in the plant, which indicates that the miPEP is taken up by the plant.

[0106] In one embodiment, the invention relates to the use as defined above, wherein the plant is treated with a composition comprising 10 -9< M to 10 -4< M of said miPEP, in particular 10 -9< M, 10 -8< M, 10 -7< M, 10 -6< M, 10 -5< M or 10 -4< M of said miPEP.

[0107] Preferably, the compositions have a concentration of 10 -8< M to 10 -5< M for application by watering or spraying on the plant.

[0108] In a complementary manner, more or less concentrated compositions may be envisaged for treating the plant with miPEP. For example, and in a non-limiting manner, more concentrated compositions comprising 10 -1< M to 10 -3< M, in particular 10 -2< M of miPEP, may be used in the case where the exogenously introduced miPEP is administered to the plant by spreading.

[0109] The solubility properties of miPEPs are determined in particular by their amino acid composition. Hydrophilic miPEPs can be solubilized and packaged in aqueous solutions, such as water. Hydrophobic miPEPs can be solubilized and packaged in solvents, such as organic solvents.

[0110] For plant treatment with miPEPs, organic solvents are non-toxic to plants in small quantities, i.e. they have no deleterious effect on plant development. Organic solvents may be selected, without limitation, from acetonitrile and acetic acid.

[0111] miPEPs can also be solubilized and packaged in organic solvent mixtures, such as a mixture of acetonitrile and acetic acid. In particular, miPEPs can be solubilized in a solution comprising 50% acetonitrile, 10% acetic acid, and 40% water (volume / volume / volume).

[0112] In particular, miPEP172c is solubilized in a solution comprising 40% water, 50% acetonitrile, 10% acetic acid (volume / volume / volume) or in water.

[0113] In particular, miPEP167c is solubilized in water.

[0114] In one embodiment, the invention relates to the use as defined above, wherein said miPEP is introduced into the plant via a nucleic acid encoding said miPEP, said nucleic acid being introduced into the plant.

[0115] In one embodiment, the invention relates to the use as defined above, in which the number of nodules is increased in the plant into which said miPEP has been introduced compared to the number of nodules in an identical plant of the same age into which no miPEP has been introduced, or compared to the number of nodules in an identical plant of the same age into which said miPEP has not been introduced.

[0116] In one embodiment, the invention relates to the use as defined above, in which the nitrogen concentration is increased in the aerial parts of the plant into which said miPEP has been introduced compared to the nitrogen concentration in the aerial parts of an identical plant of the same age into which no miPEP has been introduced, or compared to the nitrogen concentration in the aerial parts of an identical plant of the same age into which said miPEP has not been introduced.

[0117] In one embodiment, the invention relates to the use as defined above, in which the weight of the pods is increased in the plant into which said miPEP has been introduced compared to the weight of the pods of an identical plant of the same age into which no miPEP has been introduced, or compared to the weight of the pods of an identical plant of the same age into which said miPEP has not been introduced.

[0118] The increase in parameters used to determine and quantify nodulation in the plant into which miPEP has been introduced (such as the number of nodules, nitrogen concentration or pod weight) is preferably demonstrated by comparison with an identical plant (i.e. a plant of the same species and / or variety), of the same age and grown under the same conditions but into which no miPEP has been introduced.

[0119] The invention also relates to the use of a miPEP introduced exogenously into a plant to promote nodulation between said plant and a bacterium, said miPEP being encoded by the primary transcript, artificially introduced into the plant, of a miR, said primary transcript, said miR and said miPEP being naturally absent in the plant, said miPEP being capable of modulating the accumulation of said miR in said plant, which miR regulates the expression of at least one gene involved in nodulation in said plant. In a particular embodiment, said primary transcript of the miR, the miR and said miPEP are introduced into the plant using a vector.

[0120] In another aspect, the invention relates to a method for promoting nodulation between a plant and a bacterium, comprising a step of introducing a miPEP into a plant exogenously, said miPEP also being naturally present in said plant, said exogenously introduced miPEP being a peptide of 3 to 100 amino acids, in particular of 4 to 100 amino acids, the sequence of which comprises or consists of a sequence identical to that of said naturally present miPEP, which sequence of the naturally present miPEP is encoded by an open reading frame located in 5' on the primary transcript of a miR, said miPEP being capable of modulating the accumulation of said miR, which miR regulates the expression of at least one gene involved in nodulation in said plant, the sum of the quantity of said exogenously introduced miPEP and that of said naturally present miPEP being strictly greater than the quantity of said naturally present miPEP.

[0121] In one embodiment, the invention relates to a method as defined above in which said gene involved in nodulation encodes a transcription factor of the AP2 family.

[0122] In one embodiment, the invention relates to a method as defined above, wherein said gene involved in nodulation is the transcription factor NNC1.

[0123] In one embodiment, the invention relates to a method as defined above, wherein said gene involved in nodulation is the gene NSP1.

[0124] In one embodiment, the invention relates to a method as defined above, wherein said gene involved in nodulation is the gene NIN.

[0125] In one embodiment, the invention relates to a method as defined above, wherein said gene involved in nodulation is the gene ENOD40-1.

[0126] In one embodiment, the invention relates to a method as defined above, wherein said gene involved in nodulation is the gene Hb2.

[0127] In one embodiment, the invention relates to a method as defined above, wherein said gene involved in nodulation is the gene nifH.

[0128] In one embodiment, the invention relates to a method as defined above, wherein said miRNA is miR172c, in particular, wherein said miR172c has a nucleotide sequence comprising or consisting of SEQ ID NO: 1.

[0129] In one embodiment, the invention relates to a method as defined above, wherein said miPEP is miPEP172c, in particular, wherein said miPEP172c has an amino acid sequence comprising or consisting of SEQ ID NO: 2.

[0130] In one embodiment, there is described a method as defined above, wherein said miRNA is miR167c, in particular, wherein said miR167c has a nucleotide sequence comprising or consisting of SEQ ID NO: 6.

[0131] In one embodiment, there is described a method as defined above, wherein said miPEP is miPEP167c, in particular, wherein said miPEP167c has an amino acid sequence comprising or consisting of SEQ ID NO: 7.

[0132] In one embodiment, the invention relates to a method as defined above, wherein said plant is a leguminous plant, such as Lotus (Lotus sp.) soybeans (Glycine max), peanut (Arachis hypogaea), the bean (Phaseolus vulgaris), the pea (Pea sativum), the lens (Culinary lens), chickpea (Cicer arietinum), broad bean and field bean (Vetica bean ), vetches (Vice sp.), vetch (Lathyrus sp.), alfalfa (Medicine sp.), clover (Clover sp.), lupin (Lupine sp.), mung bean ( Radiata vine), licorice (Glycyrrhiza glabra), rosewood (Dalbergia), the birdsfoot trefoil (Lotus corniculatus), sainfoin (Onobrychis viciifolia ), rooibos (Aspalathus linearis), fenugreek (Trigonella fenugreek ).

[0133] In one embodiment, the invention relates to a method as defined above, wherein said plant is sugar beet ( Beetroot ).

[0134] In one embodiment, the invention relates to a method as defined above, wherein said bacterium is a bacterium of the family of Rhizobiaceae.

[0135] In a particular embodiment, the invention relates to a method as defined above, in which said bacterium is chosen from the genera Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium Or Azorhizobium.

[0136] In one embodiment, the invention relates to a method as defined above, wherein said bacterium is a bacterium selected from Rhizobium leguminosarum, Rhizobium etli, Rhizobium tropici, Rhizobium galegae, Sinorhizobium sp. NGR234, Sinorhizobium meliloti, Sinorhizobium fredii, Sinorhizobium saheli, Sinorhizobium teranga, Mesorhizobium ciceri, Mesorhizobium huakuii, Mesorhizobium loti, Bradyrhizobium elkanii, Bradyrhizobium japonicum, Bradyrhizobium lupini, Bradyrhizobium sp. "cowpea'' and Azorhizobium caulinodans.

[0137] In one embodiment, the invention relates to a method as defined above, for promoting nodulation between a plant Glycine maxand a bacterium Bradyrhizobium japonicum, wherein miPEP172c is introduced exogenously into said plant Glycine max, said miPEP172c also being naturally present in said plant Glycine max, said exogenously introduced miPEP172c being a peptide comprising or consisting of a sequence identical to that of said naturally occurring miPEP172c, which naturally occurring miPEP172c is a peptide of 3 to 100 amino acids whose sequence is encoded by an open reading frame located 5' on the primary transcript of miR172c, said miPEP172c being capable of increasing the accumulation of said miR172c, which miR172c regulates the expression of at least one gene involved in nodulation in Glycine max, the sum of the quantity of said miPEP172c introduced exogenously and that of said miPEP172c naturally present being strictly greater than the quantity of said miPEP 172c naturally present.

[0138] In one embodiment, there is described a method as defined above, for promoting nodulation between a plant Glycine max and a bacterium Bradyrhizobium japonicum, wherein miPEP167c is introduced exogenously into said plant Glycine max, said miPEP167c also being naturally present in said plant Glycine max, said exogenously introduced miPEP167c being a peptide comprising or consisting of a sequence identical to that of said naturally occurring miPEP167c, which naturally occurring miPEP167c is a peptide of 3 to 100 amino acids whose sequence is encoded by an open reading frame located 5' on the primary transcript of miR167c, said miPEP167c being capable of increasing the accumulation of said miR167c, which miR167c regulates the expression of at least one gene involved in nodulation in Glycine max,the sum of the quantity of said miPEP167c introduced exogenously and that of said miPEP167c naturally present being strictly greater than the quantity of said miPEP167c naturally present.

[0139] In one embodiment, the invention relates to a method as defined above, in which said miPEP is introduced externally into the plant, preferably by watering, by spraying or by adding a fertilizer, a potting soil, a growing substrate or using a support in contact with the plant.

[0140] In one embodiment, the invention relates to a method as defined above, in which said miPEP is introduced externally into a plant, by the addition of granules.

[0141] In one embodiment, the invention relates to a method as defined above, in which said miPEP is introduced externally into a seed or a seed, preferably by watering, by spraying or by adding a fertilizer, a potting soil, a growing substrate or using a support in contact with the seed or the seed.

[0142] In one embodiment, the invention relates to the method as defined above, wherein said miPEP is used to treat the plant in seed form.

[0143] In one embodiment, the invention relates to a method as defined above, wherein said miPEP is 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.

[0144] In one embodiment, the invention relates to a method as defined above, wherein said miPEP is introduced into the plant via a nucleic acid encoding said miPEP, said nucleic acid being introduced into the plant.

[0145] In one embodiment, the invention relates to a method as defined above, in which the number of nodules is increased in the plant into which said miPEP has been introduced compared to the number of nodules in an identical plant of the same age into which no miPEP has been introduced, or compared to the number of nodules in an identical plant of the same age into which said miPEP has not been introduced.

[0146] In one embodiment, the invention relates to a method as defined above, in which the nitrogen concentration is increased in the aerial parts of the plant into which said miPEP has been introduced compared to the nitrogen concentration in the aerial parts of an identical plant of the same age into which no miPEP has been introduced, or compared to the nitrogen concentration in the aerial parts of an identical plant of the same age into which said miPEP has not been introduced.

[0147] In one embodiment, the invention relates to a method as defined above, in which the weight of the pods is increased in the plant into which said miPEP has been introduced compared to the weight of the pods of an identical plant of the same age into which no miPEP has been introduced, or compared to the weight of the pods of an identical plant of the same age into which said miPEP has not been introduced.

[0148] In another aspect, the invention relates to a method of producing a transgenic plant comprising: a) a step of introducing a nucleic acid encoding a miPEP of 3 to 100 amino acids, in particular of 4 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 naturally present miPEP being a peptide whose sequence is encoded by an open reading frame located 5' on the primary transcript of a miR, said miPEP being capable of modulating the accumulation of said miR in the plant, which miR regulates the expression of at least one gene involved in nodulation, and b) a step of culturing the plant, or at least one cell of said plant, obtained in step a) under conditions allowing the production of a transgenic plant.

[0149] In one embodiment, the invention relates to a method for producing a transgenic plant as defined above, wherein said transgenic plant obtained in step b) is more capable of nodulation compared to an identical plant into which said nucleic acid has not been introduced.

[0150] In one embodiment, the invention relates to a method of producing a transgenic plant as defined above, wherein expression of said miPEP encoded by the nucleic acid introduced into the plant results in improved nodulation compared to an identical plant into which said nucleic acid has not been introduced.

[0151] In one embodiment, the invention relates to a method for producing a transgenic plant as defined above, wherein step a) is carried out using a vector containing said nucleic acid, preferably a plasmid.

[0152] In one embodiment, the invention relates to a method for producing a transgenic plant as defined above, wherein said nucleic acid does not comprise the complete sequence of said miR.

[0153] In one embodiment, the invention relates to a method for producing a transgenic plant as defined above, in which the expression of said nucleic acid of step a) is placed under the control of a strong promoter, preferably a strong constitutive promoter such as the 35S promoter.

[0154] In one embodiment, the invention relates to a method for producing a transgenic plant as defined above, wherein said gene involved in nodulation encodes a transcription factor of the AP2 family.

[0155] In one embodiment, the invention relates to a method for producing a transgenic plant as defined above, wherein said gene involved in nodulation is an endogenous gene and encodes a transcription factor of the AP2 family.

[0156] In one embodiment, the invention relates to a method for producing a transgenic plant as defined above, wherein said gene involved in nodulation encodes the transcription factor NNC1.

[0157] In one embodiment, the invention relates to a method for producing a transgenic plant as defined above, wherein said gene involved in nodulation is an endogenous gene and encodes the transcription factor NNC1.

[0158] In one embodiment, the invention relates to a method for producing a transgenic plant as defined above, wherein said gene involved in nodulation is the gene NSP1.

[0159] In one embodiment, the invention relates to a method for producing a transgenic plant as defined above, wherein said gene involved in nodulation is an endogenous gene. NSP1.

[0160] In one embodiment, the invention relates to a method for producing a transgenic plant as defined above, wherein said gene involved in nodulation is the gene NIN.

[0161] In one embodiment, the invention relates to a method for producing a transgenic plant as defined above, wherein said gene involved in nodulation is an endogenous gene. NIN.

[0162] In one embodiment, the invention relates to a method for producing a transgenic plant as defined above, wherein said gene involved in nodulation is the gene ENOD40-1.

[0163] In one embodiment, the invention relates to a method for producing a transgenic plant as defined above, wherein said gene involved in nodulation is an endogenous gene. ENOD40-1.

[0164] In one embodiment, the invention relates to a method for producing a transgenic plant as defined above, wherein said gene involved in nodulation is the gene Hb2.

[0165] In one embodiment, the invention relates to a method for producing a transgenic plant as defined above, wherein said gene involved in nodulation is the endogenous gene Hb2.

[0166] In one embodiment, the invention relates to a method for producing a transgenic plant as defined above, wherein said gene involved in nodulation is the gene nifH.

[0167] In one embodiment, the invention relates to a method for producing a transgenic plant as defined above, wherein said gene involved in nodulation is the endogenous gene nifH.

[0168] In one embodiment, the invention relates to a method for producing a transgenic plant as defined above, wherein said miRNA is miR172c, in particular, wherein said miR172c has a nucleotide sequence comprising or consisting of SEQ ID NO: 1.

[0169] In one embodiment, the invention relates to a method for producing a transgenic plant as defined above, wherein said miPEP is miPEP172c, in particular, wherein said miPEP172c has an amino acid sequence comprising or consisting of SEQ ID NO: 2.

[0170] In one embodiment, the invention relates to a method for producing a transgenic plant as defined above, wherein said nucleic acid introduced in step a) comprises a nucleotide sequence consisting of SEQ ID NO: 3.

[0171] In one embodiment, there is described a method of producing a transgenic plant as defined above, wherein said miRNA is miR167c, in particular, wherein said miR167c has a nucleotide sequence comprising or consisting of SEQ ID NO: 6.

[0172] In one embodiment, there is described a method of producing a transgenic plant as defined above, wherein said miPEP is miPEP167c, in particular, wherein said miPEP167c has an amino acid sequence comprising or consisting of SEQ ID NO: 7.

[0173] In one embodiment, a method for producing a transgenic plant as defined above is described, wherein said nucleic acid introduced in step a) comprises a nucleotide sequence consisting of SEQ ID NO: 8.

[0174] In one embodiment, the invention relates to a method as defined above, wherein said plant is a leguminous plant, such as Lotus (Lotus sp.) soybeans ( Glycine max ), peanut (Arachis hypogaea), the bean (Phaseolus vulgaris), the pea (Pea sativum), the lens ( Culinary lens), chickpea (Cicer arietinum), broad bean and field bean (Vetica bean ), vetches (Vice sp.), vetch (Lathyrus sp.), alfalfa (Medicine sp.), clover (Clover sp.), lupin (Lupine sp.), mung bean ( Radiata vine), licorice (Glycyrrhiza glabra), rosewood (Dalbergia), the birdsfoot trefoil (Lotus corniculatus), sainfoin (Onobrychis viciifolia ), rooibos (Aspalathus linearis), fenugreek (Trigonella fenugreek ).

[0175] In one embodiment, the invention relates to a method as defined above, wherein said plant is sugar beet ( Beetroot ).

[0176] In one embodiment, the invention relates to a method as defined above, wherein said bacterium is a bacterium of the family of Rhizobiaceae.

[0177] In a particular embodiment, the invention relates to a method as defined above, in which said bacterium is chosen from the genera Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium Or Azorhizobium.

[0178] In one embodiment, the invention relates to a method as defined above, wherein said bacterium is a bacterium selected from Rhizobium leguminosarum, Rhizobium etli, Rhizobium tropici, Rhizobium galegae, Sinorhizobium sp. NGR234, Sinorhizobium meliloti, Sinorhizobium fredii, Sinorhizobium saheli, Sinorhizobium teranga, Mesorhizobium ciceri, Mesorhizobium huakuii, Mesorhizobium loti, Bradyrhizobium elkanii, Bradyrhizobium japonicum, Bradyrhizobium lupini, Bradyrhizobium sp. "cowpea'' and Azorhizobium caulinodans.

[0179] In one embodiment, the invention relates to a method of producing a transgenic plant as defined above, comprising: a) a step of introducing a nucleic acid containing the nucleotide sequence SEQ ID NO: 3, encoding miPEP172c consisting of the amino acid sequence SEQ ID NO: 2, into a plant Glycine max, or in at least one cell of said plant Glycine max, under conditions allowing the expression of miPEP172c, said miPEP172c also being naturally present in said plant Glycine max, said naturally occurring miPEP being a peptide whose sequence is encoded by an open reading frame located 5' on the primary transcript of miR172c, said miPEP172c being capable of modulating the accumulation of said miR172c, which miR172c regulates the expression of at least one gene involved in nodulation in Glycine max, And b) a step of culturing the plant, or at least one cell of said plant, obtained in step a) under conditions allowing a plant to be obtained Glycine max transgenic.

[0180] In one embodiment, there is described a method of producing a transgenic plant as defined above, comprising: a) a step of introducing a nucleic acid containing the nucleotide sequence SEQ ID NO: 8, encoding miPEP167c consisting of the amino acid sequence SEQ ID NO: 7, into a plant Glycine max, or in at least one cell of said plant Glycine max, under conditions allowing the expression of miPEP167c, said miPEP167c also being naturally present in said plant Glycine max, said naturally occurring miPEP being a peptide whose sequence is encoded by an open reading frame located 5' on the primary transcript of miR167c, said miPEP167c being capable of modulating the accumulation of said miR167c, which miR167c regulates the expression of at least one gene involved in nodulation in Glycine max, And b)a step of culturing the plant, or at least one cell of said plant, obtained in step a) under conditions allowing a plant to be obtained Glycine max transgenic.

[0181] In one embodiment, the invention relates to a production method as defined above, wherein said miPEP is introduced into the plant via a nucleic acid encoding said miPEP, said nucleic acid being introduced into the plant.

[0182] In one embodiment, the invention relates to a method for producing a transgenic plant as defined above, in which the number of nodules is increased in the plant into which said miPEP has been introduced compared to the number of nodules in an identical plant of the same age into which no miPEP has been introduced, or compared to the number of nodules in an identical plant of the same age into which said miPEP has not been introduced.

[0183] In one embodiment, the invention relates to a method for producing a transgenic plant as defined above, in which the nitrogen concentration is increased in the aerial parts of the plant into which said miPEP has been introduced compared to the nitrogen concentration in the aerial parts of an identical plant of the same age into which no miPEP has been introduced, or compared to the nitrogen concentration in the aerial parts of an identical plant of the same age into which said miPEP has not been introduced.

[0184] In one embodiment, the invention relates to a method for producing a transgenic plant as defined above, in which the weight of the pods is increased in the plant into which said miPEP has been introduced compared to the weight of the pods of an identical plant of the same age into which no miPEP has been introduced, or compared to the weight of the pods of an identical plant of the same age into which said miPEP has not been introduced.

[0185] In one aspect, the invention also relates to a transgenic plant as obtained by the production method as defined above.

[0186] In another aspect, the invention relates to a plant into which a miPEP has been introduced according to the use or method for promoting nodulation described above.

[0187] In another aspect, the invention relates to a peptide having an amino acid sequence having at least 80% identity, preferably at least 90% identity, with the amino acid sequence SEQ ID NO: 2.

[0188] In a particular embodiment, the invention relates to a peptide comprising or consisting of sequence SEQ ID NO: 2.

[0189] In a particular embodiment, said peptide of sequence SEQ ID NO: 2 is an isolated and / or purified peptide, a synthetic peptide or a recombinant peptide.

[0190] In another aspect, the invention relates to a composition, in particular a phytosanitary composition, comprising miPEP172c as an active substance, said miPEP172c preferably consisting of SEQ ID NO: 2.

[0191] In another aspect, the invention relates to a peptide having an amino acid sequence having at least 80% identity, preferably at least 90% identity, with the amino acid sequence SEQ ID NO: 7.

[0192] In a particular embodiment, the invention relates to a peptide comprising or consisting of sequence SEQ ID NO: 7.

[0193] In a particular embodiment, said peptide of sequence SEQ ID NO: 7 is an isolated and / or purified peptide, a synthetic peptide or a recombinant peptide.

[0194] In one embodiment, the invention relates to a peptide as defined above, said peptide being labeled.

[0195] In a non-limiting manner, a labeled peptide can in particular be obtained by fusing said peptide to another peptide sequence, to a fluorescent marker (GUS, GFP, LacZ, etc.), to a signal sequence (allowing the peptide to be addressed), to a Tag sequence (allowing the purification of the miPEP) or even to a radioactive isotope.

[0196] In the case where the peptide of the invention is labeled using another peptide sequence, this is not taken into account to calculate a percentage of identity with respect to the peptide of the invention.

[0197] In another aspect, there is described a composition, in particular a phytosanitary composition, comprising miPEP167c as an active substance, said miPEP167c preferably consisting of SEQ ID NO: 7.

[0198] In another aspect, the invention relates to a composition as defined above, wherein said miPEP172c is 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.

[0199] Preferably, a composition as defined above has a concentration of 10 -8< M to 10 -5< M for application by watering or spraying on the plant or by adding a fertilizer, potting soil, growing medium or using a support in contact with the plant.

[0200] Preferably, a composition as defined above has a concentration of 10 -8< M to 10 -5< M for application by watering or spraying onto the seed or by adding a fertilizer, potting soil, growing medium or using a carrier in contact with the seed or seed.

[0201] In a complementary manner, more or less concentrated compositions may be envisaged for treating the plant with miPEP. For example, and in a non-limiting manner, more concentrated compositions comprising 10 -1< M to 10 -3< M, in particular 10 -2< M of miPEP, may be used in the case where the exogenously introduced miPEP is administered to the plant by spreading.

[0202] In another aspect, the invention relates to a composition as defined above, further comprising an excipient, a diluent or a solvent.

[0203] In one embodiment, the invention relates to a composition as defined above formulated so as to form a coating.

[0204] In one embodiment, the invention relates to a composition as defined above: in the form of a granule, formulated to form a granule, or formulated to be contained in a granule.

[0205] In another aspect, the invention relates to a composition comprising in combination an amount of seeds of a plant and an amount of a peptide whose sequence comprises or consists of a sequence identical to that of a miPEP naturally present in said plant.

[0206] In one embodiment, the invention relates to a composition comprising in combination an amount of seeds of a plant, in particular Glycine max, and an amount of a peptide whose sequence comprises or consists of a sequence identical to that of miPEP172c.

[0207] In one embodiment, a composition is described comprising in combination an amount of seeds of a plant, in particular Glycine max,and an amount of a peptide whose sequence comprises or consists of a sequence identical to that of miPEP167c. In another aspect, the invention relates to a composition as defined above, further comprising an excipient, a diluent or a solvent.

[0208] In one embodiment, the invention relates to a composition as defined above formulated so as to form a coated seed or a coated grain.

[0209] The coating can be carried out using processes conventionally used in the food industry and can be obtained using a material capable of disintegrating in a solvent or in the earth, such as a binder or clay.

[0210] The composition containing said miPEP can in particular be applied to the seed, under, in or on the coating layer.

[0211] According to the invention, the coating can be used, for example, to confer particular properties to a miPEP composition, or to a seed composition in combination with a miPEP.

[0212] In another aspect, the invention relates to a seed coated with a composition comprising a miPEP capable of modulating the accumulation of a miR in the plant derived from the seed, which miR regulates the expression of at least one gene involved in nodulation in said plant.

[0213] In one embodiment, the invention relates to a seed coated with a composition comprising miPEP172c.

[0214] In one embodiment, the invention relates to a seed coated with a composition comprising a peptide whose sequence has at least 80% identity, preferably at least 90% identity with the sequence SEQ ID NO: 2.

[0215] In one embodiment, the invention relates to a seed coated with a composition comprising the peptide SEQ ID NO: 2.

[0216] In one embodiment, a seed coated with a composition comprising miPEP167c is described.

[0217] In one embodiment, a seed coated with a composition comprising a peptide whose sequence has at least 80% identity, preferably at least 90% identity with the sequence SEQ ID NO: 7 is described.

[0218] In one embodiment, a seed coated with a composition comprising the peptide SEQ ID NO: 7 is described.

[0219] In one embodiment, the invention relates to a coated seed as defined above, said plant being a leguminous plant, such as Lotus (Lotus sp.) soybeans (Glycine max), peanut (Arachis hypogaea), the bean (Phaseolus vulgaris), the pea (Pea sativum), the lens ( Culinary lens ), chickpea (Cicer arietinum), broad bean and field bean (Vetica bean ), vetches (Vice sp.), vetch (Lathyrus sp.), alfalfa (Medicine sp.), clover (Clover sp.), lupin (Lupine sp.), mung bean ( Radiata vine), licorice (Glycyrrhiza glabra), rosewood (Dalbergia), the birdsfoot trefoil (Lotus corniculatus), sainfoin (Onobrychis viciifolia ), rooibos (Aspalathus linearis), fenugreek (Trigonella fenugreek ).

[0220] In another aspect, the invention relates to a protocol for producing a recombinant peptide, the sequence of which comprises or consists of a sequence identical to that of a miPEP as defined above, comprising a step of transforming an organism with an expression vector encoding said recombinant peptide.

[0221] In one embodiment, said organism is selected from the group consisting of bacteria, yeasts, fungi (other than yeasts), animal cells, plants and animals.

[0222] In one embodiment, said organism is Escherichia coli.

[0223] In particular, the invention relates to a protocol for producing a recombinant peptide as defined above, comprising the following steps: the nucleic acid encoding said recombinant peptide is linked to a nucleic acid encoding a tag, such as GST, the expression vector containing said nucleic acid encoding said recombinant peptide is introduced into the bacterium E. coli, the bacteria E. coli containing the expression vector is cultured in LB medium preferably to an OD between 0.2 and 0.4, the production of the recombinant peptide is induced with IPTG, preferably for 4 to 5 hours, the bacteria E. coli are centrifuged and lysed, the supernatant is filtered, said recombinant peptide is purified on a glutathione sepharose affinity column, if necessary, cleave the GST with a protease.

[0224] In another aspect, the invention relates to a bacterium transformed with a sequence encoding a miPEP as defined above.

[0225] In one embodiment, the invention relates to a bacterium transformed with a sequence encoding a miPEP, said bacterium being a bacterium of the family of Rhizobiaceae. In one embodiment, the invention relates to a bacterium transformed with a sequence encoding a miPEP, said bacterium being selected from the genera Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium Or Azorhizobium.

[0226] In one embodiment, the invention relates to a bacterium transformed with a sequence encoding a miPEP, said bacterium being selected from Rhizobium leguminosarum, Rhizobium etli, Rhizobium tropici, Rhizobium galegae, Sinorhizobium sp. NGR234, Sinorhizobium meliloti, Sinorhizobium fredii, Sinorhizobium saheli, Sinorhizobium teranga, Mesorhizobium ciceri, Mesorhizobium huakuii, Mesorhizobium loti, Bradyrhizobium elkanii, Bradyrhizobium japonicum, Bradyrhizobium lupini, Bradyrhizobium sp. "cowpea'' and Azorhizobium caulinodans.

[0227] In another aspect, the invention relates to an antibody specifically recognizing miPEP172c, in particular said miPEP172c consisting of SEQ ID NO: 2.

[0228] Such an antibody may be obtained from a method known to those skilled in the art, such as for example by injecting said miPEP172c into a non-human animal to trigger an immunization reaction and the production of antibodies by said animal.

[0229] In another aspect, the invention relates to a method for immunolocalization of miPEP172c comprising a step of labeling a biological sample of a plant with an antibody specifically recognizing said miPEP172c.

[0230] In another aspect, there is described an antibody specifically recognizing miPEP167c, in particular said miPEP167c consisting of SEQ ID NO: 7.

[0231] Such an antibody may be obtained from a method known to those skilled in the art, such as for example by injecting said miPEP167c into a non-human animal to trigger an immunization reaction and the production of antibodies by said animal.

[0232] In another aspect, a method for immunolocalizing miPEP167c is described comprising a step of labeling a biological sample of a plant with an antibody specifically recognizing said miPEP167c.

[0233] In another aspect, the invention relates to a method of culturing bacteria, comprising a step of bringing said bacteria into contact: with a mixture comprising a plant or a part of a plant, in particular a root culture, and a peptide whose sequence comprises or consists of a sequence identical to that of a miPEP naturally present in said plant, said naturally present miPEP being a peptide whose sequence is encoded by an open reading frame located 5' on the primary transcript of a miR, said miPEP being capable of modulating the accumulation of said miR, which miR regulates the expression of at least one gene involved in nodulation in said plant, or with a transgenic plant as defined above, the plant, the plant part, and the transgenic plant being capable of forming a symbiosis with said bacterium.

[0234] In a particular embodiment, the invention relates to the method as defined above in which the cultured bacteria belong to the family of Rhizobiaceae.

[0235] In a particular embodiment, the invention relates to the method as defined above in which said bacteria are chosen from the genera Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium Or Azorhizobium.

[0236] In a particular embodiment, the invention relates to the method as defined above in which said bacteria are chosen from Rhizobium leguminosarum, Rhizobium etli, Rhizobium tropici, Rhizobium galegae, Sinorhizobium sp. NGR234, Sinorhizobium meliloti, Sinorhizobium fredii, Sinorhizobium saheli, Sinorhizobium teranga, Mesorhizobium ciceri, Mesorhizobium huakuii, Mesorhizobium loti, Bradyrhizobium elkanii, Bradyrhizobium japonicum, Bradyrhizobium lupini, Bradyrhizobium sp. "cowpea'' and Azorhizobium caulinodans.

[0237] In particular, the method of culturing said bacteria as defined above is carried out under culture conditions allowing the growth, or even the improvement of the growth of the plant, the part of the plant, and the transgenic plant, and that of the bacteria.

[0238] In particular, the process of culturing bacteria as defined above is carried out under culture conditions allowing symbiosis between the bacteria and the plant, plant part or transgenic plant.

[0239] In one embodiment, the peptide present in the mixture is an isolated peptide, an isolated and / or purified peptide, a synthetic peptide or a recombinant peptide.

[0240] In one embodiment, the invention relates to a method for culturing bacteria as defined above, wherein said miR is miR172c, in particular, wherein said miR172c has a nucleotide sequence consisting of SEQ ID NO: 1.

[0241] In one embodiment, the invention relates to a method of culturing bacteria as defined above, wherein said miPEP is miPEP172c, in particular, wherein said miPEP172c has an amino acid sequence consisting of SEQ ID NO: 2.

[0242] In one embodiment, there is described a method of culturing bacteria as defined above, wherein said miR is miR167c, in particular, wherein said miR167c has a nucleotide sequence consisting of SEQ ID NO: 6.

[0243] In one embodiment, there is described a method of culturing bacteria as defined above, wherein said miPEP is miPEP167c, in particular, wherein said miPEP167c has an amino acid sequence consisting of SEQ ID NO: 7.

[0244] In particular, the invention relates to a method of culturing bacteria as defined above, wherein said plant part is a root or a root fragment.

[0245] In another aspect, the invention relates to a method for producing inoculum of bacteria, in particular bacteria of the family of Rhizobiaceae, including: a step of co-cultivation of bacteria with a living plant material, called host plant, corresponding at least in part to a constituent part of the root of a plant capable of forming a symbiosis with said bacteria, and a step of bringing a quantity of a peptide into contact with the above-mentioned co-culture, said peptide having a sequence comprising or consisting of a sequence identical to that of a miPEP naturally present in said host plant, said naturally present miPEP being a peptide whose sequence is encoded by an open reading frame located 5' on the primary transcript of a miR, said miPEP being capable of modulating the accumulation of said miR, which miR regulates the expression of at least one gene involved in nodulation in said host plant.

[0246] The invention also relates to a method for producing inoculum of bacteria, in particular bacteria of the family of Rhizobiaceae,comprising a step of co-cultivation of the bacteria with a living plant material, called a host plant, corresponding at least in part to a constituent part of the root of a plant capable of forming a symbiosis with said bacteria, and said host plant being a transgenic plant or a plant into which a peptide has been introduced, said peptide having a sequence comprising or consisting of a sequence identical to that of a miPEP naturally present in said host plant, said naturally present miPEP being a peptide whose sequence is encoded by an open reading frame located 5' on the primary transcript of a miR, said miPEP being capable of modulating the accumulation of said miR, which miR regulates the expression of at least one gene involved in nodulation in said host plant.

[0247] The invention also relates to a method for producing inoculum of bacteria, in particular bacteria of the family of Rhizobiaceae,comprising a mixing step bringing together: bacteria, a living plant material, called a host plant, corresponding at least in part to a constituent part of the root of a plant capable of forming a symbiosis with said bacteria, and a peptide, said peptide having a sequence comprising or consisting of a sequence identical to that of a miPEP naturally present in said host plant, said naturally present miPEP being a peptide whose sequence is encoded by an open reading frame located 5' on the primary transcript of a miR, said miPEP being capable of modulating the accumulation of said miR, which miR regulates the expression of at least one gene involved in nodulation in said host plant.

[0248] In another aspect, the invention relates to a bacterial inoculum, in particular an inoculum of bacteria of the family of Rhizobiaceae,suitable for inoculation of a host plant, comprising at least one bacterium and a peptide whose sequence comprises or consists of a sequence identical to that of a miPEP naturally present in the host plant, said miPEP naturally present in the host plant being a peptide whose sequence is encoded by an open reading frame located 5' on the primary transcript of a miR, said miPEP being capable of modulating the accumulation of said miR, which miR regulates the expression of at least one gene involved in nodulation in said host plant.

[0249] Preferably, said miPEP is miPEP172c.

[0250] Preferably, said miPEP is miPEP167c.

[0251] The peptide used to produce the inoculum, or the peptide present in the inoculum, is in particular an isolated peptide, an isolated and / or purified peptide, a synthetic peptide or a recombinant peptide.

[0252] Preferably, the inoculum additionally contains a plant or plant part, in particular a root, root culture or root part.

[0253] The sequences of miPEP172c, its open reading frame, miR172c and the primary transcript of miR172c in Glycine max are shown in Table 1. Table 1. miR172c GGAAUCUUGAUGAUGCUGCAG SEQ ID NO: 1 miPEP172c MWVLCLFCWPTYTHGS SEQ ID NO: 2 miORF172c SEQ ID NO: 3 pri-miR172c SEQ ID NO: 4

[0254] The sequences of miPEP167c, its open reading frame, miR167c and the primary transcript of miR167c in Glycine max are shown in Table 2. Table 2. miR167c UGAAGCUGCCAGCAUGAUCUG SEQ ID NO: 6 miPEP167c MKGVHHFFHHKYVGLRG SEQ ID NO: 7 miORF167c SEQ ID NO: 8 pri-miR167c SEQ ID NO: 9

[0255] Pages 36 to 50 correspond to extracts from French patent application no. FR 13 / 60727 filed on October 31, 2013 for “ Micropeptides and their use to modulate gene expression »

[0256] The subject of the requestFR 13 60727 concerns micropeptides (peptides encoded by microRNAs or "miPEPs") and their use to modulate gene expression.

[0257] MicroRNAs (miRs) are small non-coding RNAs, approximately 21 nucleotides in length after maturation, that control the expression of target genes at the post-transcriptional level, by degrading the target mRNA or inhibiting its translation. MiRs are found in plants and animals.

[0258] Target genes are often key genes in developmental processes. For example, they encode transcription factors or proteasome proteins.

[0259] The regulation of miR expression is very little known, but we do know that, like most coding genes, it involves an RNA polymerase II: this enzyme produces a primary transcript, called "pri-miR", which is then processed by a protein complex containing Dicer-type enzymes. This processing first leads to the formation of a miR precursor called "pre-miR", which has a secondary stem-loop structure containing the miR and its complementary miR* sequence. The precursor is then processed, leading to the formation of a shorter double-stranded RNA containing the miR and the miR*. The miR is then taken over by the RISC complex, which cleaves the mRNA of the target gene or inhibits its translation.

[0260] Furthermore, it has been shown that the presence of introns in the primary transcript of microRNA increases the expression of mature microRNA ( Schwab et al., EMBO Rep., 14(7):615-21, 2013 ). However, due to experimental difficulties, primary microRNA transcripts, or pri-miRs, are very little studied.

[0261] Approximately 50% of eukaryotic genes have, within their 5'UTR (5' Untranslated Region) upstream of the coding sequence, small open reading frames. These small open reading frames (or "uORFs" for upstream ORFs) can play a role in regulating translation, mainly in cis, by modulating the fixation and speed of ribosomes on the mRNA, but also in trans according to a mechanism still unknown, via peptides encoded by said uORFs ( Combier et al., Gene Dev, 22:1549-1559, 2008 ). By definition, uORFS are present upstream of coding genes.

[0262] Recently, small ORFs have also been discovered in long intergenic non-coding RNAs (lincRNAs) whose putative function, if any, is not known ( Ingolia et al., Cell, 147(4):789-802, 2011 ; Guttman & Rinn, Nature, 482(7385):339-46, 2012 ).

[0263] However, no examples have yet been reported concerning the existence of ORFs encoding peptides within non-coding microRNAs. Until now, microRNAs, and by extension their primary transcript, have always been considered, due to their particular mode of action, as non-coding regulatory RNAs that do not produce any peptides.

[0264] One aspect of the subject matter of the application FR 13 60727 is to propose peptides capable of modulating the expression of microRNAs.

[0265] Another aspect of the subject of the request FR 13 60727 is to propose a means of modulating the expression of one or more target genes of a microRNA.

[0266] The purpose of the request FR 13 60727 has the advantage of allowing easier and more efficient control of the expression of genes targeted by microRNAs, using a means other than microRNA.

[0267] The purpose of the request FR 13 60727 thus relates to a method for detecting and identifying a micropeptide (miPEP) coded by a nucleotide sequence contained in the sequence of the primary transcript of a microRNA, including: a) a step of detecting an open reading frame of 15 to 303 nucleotides contained in the sequence of the primary transcript of said microRNA, then b) a comparison step between: the accumulation of said microRNA in a given eukaryotic cell expressing said microRNA, in the presence of a peptide encoded by a nucleotide sequence identical to or degenerate from that of said open reading frame, said peptide being present in the cell independently of the transcription of the primary transcript of said microRNA, and the accumulation of said microRNA in a eukaryotic cell of the same type as the above-mentioned determined eukaryotic cell expressing said microRNA, in the absence of said peptide, wherein, a modulation of the accumulation of said microRNA in the presence of said peptide relative to the accumulation of said microRNA in the absence of said peptide indicates the existence of a micropeptide encoded by said open reading frame.

[0268] In a first step, the process of detecting and identifying a micropeptide therefore consists of detecting on the primary transcript of a microRNA the existence of an open reading frame potentially coding for a peptide.

[0269] The second step allows the characterization of said peptide, that is to say, to determine whether said peptide corresponds to a peptide actually produced in the cell, by looking for an effect of said peptide on the accumulation of said microRNA.

[0270] To demonstrate an effect of the peptide on microRNA accumulation, a large amount of peptide is introduced into a first cell expressing said microRNA. The accumulation of microRNA in this first cell is then measured and compared with the accumulation of microRNA in a second cell identical to the first, but not containing said peptide.

[0271] The observation of a variation in the quantities of microRNA between cells in the presence and absence of the peptide thus indicates (i) that there is a peptide encoded on the primary transcript of said microRNA, (ii) that the sequence of this peptide is encoded by the open reading frame identified on the primary transcript of said microRNA, and (iii) that said peptide acts on the accumulation of said microRNA.

[0272] The purpose of the request FR 13 60727 is therefore based on the unexpected double observation made by the Inventors that on the one hand, there are open reading frames capable of coding micropeptides present on the primary transcripts of microRNAs, and on the other hand that said micropeptides are capable of modulating the accumulation of said microRNAs.

[0273] In the application FR 13 60727 , the terms "microRNA", "non-coding microRNA" and "miR" are equivalent and can be used interchangeably. They define small RNA molecules of approximately 21 nucleotides, which are not translated and do not lead to a peptide or protein.

[0274] However, in this mature form, microRNAs perform a regulatory function for certain genes via post-transcriptional mechanisms, such as through the RISC complex.

[0275] The primary transcript of the microRNA or "pri-miR" corresponds to the RNA molecule directly obtained from the transcription of the DNA molecule. Generally, this primary transcript undergoes one or more post-transcriptional modifications, which result, for example, in a particular structure of the RNA or cleavage of certain parts of the RNA by splicing phenomena, and which lead to the precursor form of the microRNA or "pre-miR", then to the mature form of the microRNA or "miR".

[0276] The terms "micro peptides" and "miPEPs" (microRNA encoded PEPtides) are equivalent and can be used interchangeably. They define a peptide that is encoded by an open reading frame present on the primary transcript of a microRNA, and which is capable of modulating the accumulation of said microRNA. Micropeptides within the meaning of the application FR 13 60727 should not be understood as necessarily being small peptides, as “micro” does not correspond to the size of the peptide.

[0277] Given the degeneracy of the genetic code, the same micropeptide can be encoded by several nucleotide sequences. Such nucleotide sequences, which differ from each other by at least one nucleotide but encode the same peptide, are called "degenerate sequences".

[0278] The terms "open reading frame" or "ORF" are equivalent and can be used interchangeably. They correspond to a sequence of nucleotides in a DNA or RNA molecule that can potentially code for a peptide or protein: the open reading frame begins with a start codon, followed by a series of codons, and ends with a stop codon.

[0279] In the application FR 13 60727 , ORFs can be specifically referred to as “miORFs” when they are present on primary microRNA transcripts.

[0280] In the application FR 13 60727 , by "accumulation" we mean the production of a molecule, such as a microRNA or a micropeptide, in the cell.

[0281] Thus, the “modulation” of the accumulation of a molecule in a cell corresponds to a modification of the quantity of this molecule present in the cell.

[0282] In one embodiment, the subject matter of the application FR 13 60727 relates to a method for detecting and identifying a miPEP as defined above, wherein the modulation of the accumulation of said microRNA is a decrease or an increase in the accumulation of said microRNA, in particular an increase.

[0283] A "decrease in accumulation" corresponds to a decrease in the quantity of said molecule in the cell.

[0284] Conversely, an “increase in accumulation” corresponds to an increase in the quantity of said molecule in the cell.

[0285] In an advantageous embodiment, the subject matter of the application FR 13 60727 relates to a method for detecting and identifying a miPEP as defined above, wherein the modulation of the accumulation of said microRNA is an increase in the accumulation of said microRNA.

[0286] In one embodiment, the subject matter of the application FR 13 60727 relates to a method for detecting and identifying a miPEP as defined above, in which the presence of said peptide in the cell results from: the introduction into the cell of a nucleic acid encoding said peptide, or of the introduction into the cell of said peptide.

[0287] In order to characterize a miPEP, it is necessary to have a cellular model expressing a microRNA and in which the said peptide to be tested is present. To do this, it is possible to introduce a peptide into the cell, either by putting the cell in contact with the said peptide, or by introducing into the cell a nucleic acid encoding the said peptide, which nucleic acid will then be translated into a peptide inside the cell.

[0288] In one embodiment, the subject matter of the application FR 13 60727 relates to a method for detecting and identifying a miPEP as defined above, wherein said open reading frame of step a) is contained in the 5' or 3' portion of said primary microRNA transcript, preferably in the 5' portion.

[0289] The 5' or 3' portions of the primary microRNA transcript correspond to the terminal portions of the RNA molecule that are cleaved during microRNA maturation. In one embodiment, the subject matter of the application FR 13 60727 relates to a method for detecting and identifying a miPEP as defined above, in which said microRNA is present in a wild-type plant cell.

[0290] In the application FR 13 60727 , a wild plant cell corresponds to a plant cell that has not been genetically modified by humans.

[0291] In one embodiment, the subject matter of the application FR 13 60727 relates to a method for detecting and identifying a miPEP as defined above, in which said determined eukaryotic cell, and said eukaryotic cell of the same type as said determined eukaryotic cell, used in step b, are plant cells, preferably cells of Medicago truncatula or Arabidopsis thaliana.

[0292] In the method of detecting and identifying a micropeptide as defined above, after having identified an ORF capable of encoding a peptide on the primary transcript of a microRNA, it is necessary to have a cellular model possessing said microRNA and said peptide, in order to be able to demonstrate a possible effect of the peptide on said microRNA.

[0293] Two options are therefore possible: the cellular model in which the miORF was identified and the one in which the effect of the peptide on the miR is demonstrated are identical, or The cellular model in which the miORF was identified and the one in which the effect of the peptide on the miR is highlighted are different.

[0294] In the first option, the cellular model used to observe an effect of the peptide is the same as the one in which the primary transcript of said microRNA was isolated. In this cellular model, the determined eukaryotic cells naturally contain said microRNA and only the peptide to be tested must be introduced into these cells. In this context, said microRNA is called "endogenous origin" because it exists naturally in cells. However, in a cell, other copies of a microRNA of endogenous origin can be added, for example by introducing into the cell a vector encoding said microRNA of endogenous origin.

[0295] In the second option, the cellular model used to observe an effect of the peptide is different from the one in which the primary transcript of said microRNA was isolated. In this cellular model, the determined eukaryotic cells contain neither the microRNA nor the peptide to be tested. Both elements must therefore be introduced into these cells. In this context, said microRNA is described as "exogenous in origin" because it does not exist naturally in cells.

[0296] In one embodiment, the subject matter of the application FR 13 60727 relates to a method for detecting and identifying a miPEP as defined above, in which said microRNA is of endogenous origin in said eukaryotic cell and in said eukaryotic cell of the same type as the above-mentioned determined eukaryotic cell, used in step b).

[0297] In one embodiment, the subject matter of the application FR 13 60727 relates to a method for detecting and identifying a miPEP as defined above in which said microRNA is of exogenous origin in said eukaryotic cell and in said eukaryotic cell of the same type as the above-mentioned determined eukaryotic cell, used in step b), said eukaryotic cells containing a vector allowing the expression of said microRNA.

[0298] In one embodiment, the subject matter of the application FR 13 60727 relates to a method for detecting and identifying a miPEP as defined above, in which the accumulation of said microRNA is determined by implementing quantitative RT-PCR or Northern blotting.

[0299] In one embodiment, the subject matter of the application FR 13 60727 relates to a method for detecting and identifying a miPEP as defined above, in which the accumulation of said microRNA is determined by implementing a DNA or RNA chip.

[0300] The accumulation of said microRNA can be determined using molecular biology techniques allowing the dosage of specific nucleic acid molecules.

[0301] In another aspect, the subject matter of the application FR 13 60727 also relates to a method for detecting and identifying a microRNA whose primary transcript sequence contains a nucleotide sequence encoding a miPEP, including: a) a step of detecting an open reading frame of 15 to 303 nucleotides contained in the sequence of the primary transcript of said microRNA, then b) a comparison step between: the accumulation of said microRNA in a given eukaryotic cell expressing said microRNA, in the presence of a peptide encoded by a nucleotide sequence identical to or degenerate from that of said open reading frame, said peptide being present in the cell independently of the transcription of the primary transcript of said microRNA, and the accumulation of said microRNA in a eukaryotic cell, of the same type as the aforementioned determined eukaryotic cell expressing said microRNA, in the absence of said peptide, wherein, a modulation of the accumulation of said microRNA in the presence of said peptide relative to the accumulation of said microRNA in the absence of said peptide indicates the existence of a microRNA whose primary transcript contains a nucleotide sequence encoding a micropeptide.

[0302] In one embodiment, the subject matter of the application FR 13 60727 relates to a method for detecting and identifying a microRNA as defined above, wherein the modulation of the accumulation of said microRNA is a decrease or an increase in the accumulation of said microRNA, in particular an increase.

[0303] In one embodiment, the subject matter of the application FR 13 60727 relates to a method for detecting and identifying a microRNA as defined above, in which the presence of said peptide in the cell results from: the introduction into the cell of a nucleic acid encoding said peptide, or of the introduction into the cell of said peptide.

[0304] In one embodiment, the subject matter of the application FR 13 60727 relates to a method for detecting and identifying a microRNA as defined above, wherein said open reading frame of step a) is contained in the 5' or 3' portion of said primary transcript of the microRNA, preferably in the 5' portion.

[0305] In one embodiment, the subject matter of the application FR 13 60727 relates to a method for detecting and identifying a microRNA as defined above, wherein said microRNA is present in a wild-type plant cell.

[0306] In one embodiment, the subject matter of the application FR 13 60727 relates to a method for detecting and identifying a microRNA as defined above, in which said eukaryotic cell, and said eukaryotic cell of the same type as the above-mentioned determined eukaryotic cell, used in step b) are plant cells, preferably Medicago truncatula cells.

[0307] In one embodiment, the subject matter of the application FR 13 60727 relates to a method for detecting and identifying a microRNA as defined above, in which said microRNA is of endogenous origin in said eukaryotic cell and in said eukaryotic cell of the same type as the above-mentioned determined eukaryotic cell, used in step b).

[0308] In one embodiment, the subject matter of the application FR 13 60727 relates to a method for detecting and identifying a microRNA as defined above in which said microRNA is of exogenous origin in said eukaryotic cell and in said eukaryotic cell of the same type as the above-mentioned determined eukaryotic cell, used in step b), said eukaryotic cells containing a vector allowing the expression of said microRNA.

[0309] In one embodiment, the subject matter of the application FR 13 60727 relates to a method for detecting and identifying a microRNA as defined above, in which the accumulation of said microRNA is determined by implementing a quantitative RT-PCR or a Northern blot.

[0310] In one embodiment, the subject matter of the application FR 13 60727 relates to a method for detecting and identifying a microRNA as defined above, in which the accumulation of said microRNA is determined by implementing a DNA or RNA chip.

[0311] In another aspect, the subject matter of the application FR 13 60727 relates to a miPEP as obtained by implementing the method as defined above.

[0312] Another aspect of the subject of the request FR 13 60727 also relates to a miPEP of 4 to 100 amino acids, preferably of 4 to 40 amino acids, encoded by a nucleotide sequence contained in the primary transcript of a microRNA, said miPEP being capable of modulating the accumulation of said microRNA in a eukaryotic cell.

[0313] Furthermore, it is worth noting that multiple miORFS can be identified on the primary transcript of a microRNA, indicating that a primary microRNA transcript can potentially encode multiple miPEPs.

[0314] It should also be noted that the effect of a miPEP is usually specific to a single microRNA, namely the one resulting from the primary transcript encoding said miPEP.

[0315] In one embodiment, the subject matter of the application FR 13 60727 relates to a miPEP as defined above, said nucleotide sequence being contained in the 5' or 3' part of said primary transcript of a microRNA, preferably in the 5' part.

[0316] In one embodiment, the subject matter of the application FR 13 60727 relates to a miPEP as defined above, said nucleotide sequence corresponding to the first open reading frame present on said primary transcript of a microRNA.

[0317] In one embodiment, the subject matter of the application FR 13 60727 relates to a miPEP as defined above, said miPEP having a basic isoelectric point, preferably greater than 8.

[0318] In another aspect, the subject matter of the application FR 13 60727 relates to a nucleic acid molecule encoding a miPEP as defined above.

[0319] In another aspect, the subject of the request FR 13 60727 relates to a vector comprising at least one nucleic acid molecule as defined above.

[0320] In another aspect, the subject matter of the application FR 13 60727 also concerns the use of at least: a miPEP as defined above, a nucleic acid encoding said miPEP, or a vector containing said nucleic acid, to modulate the expression of at least one gene in a given eukaryotic cell, said determined eukaryotic cell being capable of expressing a microRNA, the primary transcript of which contains at least one nucleotide sequence coding said at least one miPEP and the accumulation of which is modulated by said at least one miPEP, the expression of said at least one gene being regulated by said microRNA.

[0321] In another aspect, the subject matter of the application FR 13 60727 also concerns the use of at least: a miPEP of 4 to 100 amino acids, preferably of 4 to 40 amino acids, encoded by a nucleotide sequence contained in the primary transcript of a microRNA, said miPEP being capable of modulating the accumulation of said microRNA in a eukaryotic cell, a nucleic acid encoding said miPEP, or a vector containing said nucleic acid, to modulate the expression of at least one gene in a given eukaryotic cell, said determined eukaryotic cell being capable of expressing a microRNA, the primary transcript of which contains at least one nucleotide sequence coding said at least one miPEP and the accumulation of which is modulated by said at least one miPEP, the expression of said at least one gene being regulated by said microRNA.

[0322] The purpose of the request FR 13 60727 is based on the surprising observation made by the Inventors that it is possible to modulate the expression of one or more target genes of the same microRNA by modulating the accumulation of said microRNA using a miPEP.

[0323] In one embodiment, the subject matter of the application FR 13 60727 relates to the use as defined above in which said determined eukaryotic cell is a plant cell.

[0324] In one embodiment, the subject matter of the application FR 13 60727 relates to the use as defined above in which said microRNA and said gene are of endogenous origin in said determined eukaryotic cell.

[0325] In one embodiment, the subject matter of the application FR 13 60727 relates to the use as defined above in which said microRNA and said gene are of exogenous origin in said determined eukaryotic cell, said determined eukaryotic cell containing at least one vector allowing the expression of said microRNA and said gene.

[0326] In the application FR 13 60727 , the expressions “of endogenous origin” and “of exogenous origin” are used to distinguish said microRNAs and / or genes from different species, given the conservation of sequences between species.

[0327] Thus, the term "endogenous in origin" indicates that the microRNA and / or the gene may be present naturally in the cell in question. Artificially, other copies of the microRNA and / or the gene of endogenous origin can nevertheless be added into the cell in question, for example by cloning.

[0328] Conversely, the term "of exogenous origin" indicates that the microRNA and / or gene are never naturally present in the cell in question. It is a microRNA and / or a gene identified in another cell type or in an organism of another species, this microRNA and / or this gene are therefore necessarily artificially introduced into the cell in question.

[0329] In the application FR 13 60727 , a genetically transformed cell can therefore contain 2 groups of microRNAs and / or genes potentially close in terms of sequence, one of endogenous origin and the other of exogenous origin.

[0330] In another aspect, the subject matter of the application FR 13 60727 relates to a method for modulating the expression of a gene regulated by a microRNA in a eukaryotic cell, comprising the implementation of a step of accumulating a miPEP in said eukaryotic cell, said miPEP having: a size of 4 to 100 amino acids, preferably 4 to 20 amino acids, and a peptide sequence identical to that encoded by a nucleotide sequence contained in the primary transcript of a microRNA regulating the expression of said gene, and being capable of modulating the accumulation of said microRNA, wherein, the accumulation of said miPEP in said eukaryotic cell induces a modulation of the expression of said gene relative to the expression of said gene without accumulation of said miPEP.

[0331] In one embodiment, the subject matter of the application FR 13 60727 relates to a method of modulating the expression of a gene as defined above, wherein the accumulation of said miPEP in the cell results from: the introduction into the cell of a nucleic acid encoding said miPEP, or of the introduction into the cell of said miPEP.

[0332] In one embodiment, the subject matter of the application FR 13 60727 relates to a method of modulating the expression of a gene as defined above in which said eukaryotic cell is a plant cell.

[0333] In one embodiment, the subject matter of the application FR 13 60727 relates to a method of modulating the expression of a gene as defined above in which said microRNA and said gene are of endogenous origin in said eukaryotic cell.

[0334] In one embodiment, the subject matter of the application FR 13 60727 relates to a method for modulating the expression of a gene as defined above in which said microRNA and said gene are of exogenous origin in said eukaryotic cell, said eukaryotic cell containing at least one vector allowing the expression of said microRNA and said gene.

[0335] In another aspect, the subject matter of the application FR 13 60727 relates to a modified eukaryotic cell containing a peptide identical to a miPEP as defined above, which peptide is present in said eukaryotic cell independently of the transcription of the primary transcript of the microRNA carrying the nucleotide sequence encoding said miPEP.

[0336] In the application FR 13 60727 , the term “modified eukaryotic cell” means that said eukaryotic cell contains a miPEP artificially introduced into the cell, whether as a peptide, or via a vector encoding said miPEP.

[0337] In one embodiment, the subject matter of the application FR 13 60727 relates to a modified eukaryotic cell as defined above, in which said microRNA is of endogenous origin.

[0338] In another embodiment, the subject matter of the application FR 13 60727 relates to a modified eukaryotic cell as defined above in which said microRNA is of exogenous origin, said modified eukaryotic cell containing a vector allowing the expression of said microRNA.

[0339] In one embodiment, the subject matter of the application FR 13 60727 relates to a modified eukaryotic cell as defined above, said cell being a plant cell.

[0340] In another aspect, the subject matter of the application FR 13 60727 relates to a plant comprising at least one modified eukaryotic cell as defined above.

[0341] In another aspect, the subject matter of the application FR 13 60727 relates to a composition comprising at least: a miPEP as defined above, a nucleic acid encoding said miPEP, or a vector containing said nucleic acid.

[0342] In another aspect, the subject matter of the application FR 13 60727 relates to a pesticide composition comprising at least: a miPEP as defined above, a nucleic acid encoding said miPEP, or a vector containing said nucleic acid.

[0343] In another aspect, the subject matter of the application FR 13 60727 relates to a phytopharmaceutical composition comprising at least : a miPEP as defined above, a nucleic acid encoding said miPEP, or a vector containing said nucleic acid.

[0344] In another aspect, the subject matter of the application FR 13 60727 relates to an eliciting composition comprising at least: a miPEP as defined above, a nucleic acid encoding said miPEP, or a vector containing said nucleic acid.

[0345] By "elicitor composition" we mean a composition capable of giving the plant a better aptitude for symbiosis or better resistance to different stresses, whether thermal, water or chemical in nature.

[0346] For this purpose, the subject of the request FR 13 60727 also concerns compositions acting on the growth (inhibition of growth or on the contrary increase of growth) and the physiology (better ability to mycorrhize, nodulate, better tolerance to different stresses) of the plant.

[0347] In another aspect, the subject matter of the application FR 13 60727 relates to a herbicidal composition comprising at least: a miPEP as defined above, a nucleic acid encoding said miPEP, or a vector containing said nucleic acid.

[0348] In another aspect, the subject matter of the application FR 13 60727 concerns a composition insecticide comprising at least: a miPEP as defined above, a nucleic acid encoding said miPEP, or a vector containing said nucleic acid.

[0349] In another aspect, the subject matter of the application FR 13 60727 relates to the use of a composition as defined above, as a herbicide for eliminating plants or slowing their growth, preferably as a herbicide specific to a species or genus of plants.

[0350] In another aspect, the subject matter of the application FR 13 60727 relates to the use of a composition as defined above, as a phytopharmaceutical agent, to promote growth and / or the development of plants, in particular for the modulation of the physiological parameters of a plant, in particular biomass, leaf area, flowering, fruit size, production and / or the selection of plant seeds, in particular for controlling the parthenocarpy or monoecy of a plant, or for modifying the physiological parameters of plant seeds, in particular germination, root establishment and resistance to water stress, or to prevent or treat plant diseases, particularly to promote resistance to infectious diseases.

[0351] In another aspect, the subject matter of the application FR 13 60727 relates to the use of a composition as defined above, to modulate the physiological parameters of a plant, in particular biomass, leaf area, or fruit size.

[0352] In one embodiment, the subject matter of the application FR 13 60727 relates to the use of a composition as defined above, for thinning orchards in order to increase the size of the fruit.

[0353] In one embodiment, the subject matter of the application FR 13 60727 relates to the use of a composition as defined above, for the production and / or the selection of plant seeds, said composition being used to control the parthenocarpy or monoecy of a plant.

[0354] In one embodiment, the subject matter of the application FR 13 60727 relates to the use of a composition as defined above, said composition being administered to said plant via the foliar route or via the root route.

[0355] In one embodiment, the subject matter of the application FR 13 60727 relates to the use of a composition as defined above, for the production and / or the selection of plant seeds.

[0356] In one embodiment, the subject matter of the application FR 13 60727 relates to the use of a composition as defined above, wherein said composition is used to modify the physiological parameters of said plant seeds, in particular root establishment, germination and resistance to water stress.

[0357] In one embodiment, the subject matter of the application FR 13 60727 relates to the use of a composition as defined above, wherein said composition is applied by coating or film-coating onto said plant seeds.

[0358] In another aspect, the subject matter of the application FR 13 60727 relates to the use of a composition as defined above, as a pesticide, for eliminating harmful organisms from plants or likely to be classified as such, particularly as an insecticide, arachnicide, limacide or rodenticide.

[0359] In one embodiment, the subject matter of the application FR 13 60727 relates to the use of a composition as defined above, as an insecticide.

[0360] In one embodiment, the subject matter of the application FR 13 60727 relates to the use of a composition as defined above, to eliminate insect pests.

[0361] In one embodiment, the subject matter of the application FR 13 60727 concerns the use of a composition as defined above, to eliminate animal species classified as harmful or likely to be classified as such, in particular muridae, notably the rat.

[0362] In another aspect, the subject matter of the application FR 13 60727 relates to the use of a composition as defined above, wherein said composition is applied to a plant to protect it from insect pests. FIGURE CAPTIONS

[0363] FIGURE 1 . Effects of treatment with miPEP172c on miR172c expression in Glycine max The y-axis indicates the relative expression of miR172c in a control plant (left column) or in a plant watered with 0.1 µM miPEP172c (right column). The error bar corresponds to the standard error of the mean (number of individuals = 9). FIGURE 2 . Effects of miPEP172c on nodule number in Glycine max The y-axis indicates the number of nodules per gram of fresh weight of root of Glycine max treated with solvent (control, left bar) or with solvent containing 0.1 µM miPEP172c (miPEP172c, right bar). The error bar corresponds to the standard error of the mean (number of individuals = 9). This experiment was repeated independently and yielded similar results. FIGURE 3 . Effects of miPEP172c on nitrogen concentration in aerial parts of Glycine max The y-axis indicates the nitrogen concentration per dry weight of leaves of Glycine max (in g / kg), treated with a solvent (control, left bar) or with a solvent containing 0.1 µM miPEP172c (miPEP172c, right bar). The error bar corresponds to the standard error of the mean (number of individuals = 9). FIGURE 4 . Effects of miPEP172c on pod fresh weight in Glycine max The y-axis indicates the fresh dry weight of the pods in Glycine max(in g), treated with a solvent (control, left bar) or with a solvent containing 0.1 µM of miPEP 172c (miPEP172c, right bar). The error bar corresponds to the standard error of the mean (number of individuals = 9). FIGURE 5 . Effects of miPEP172c on nodule number in Glycine max The y-axis indicates the number of nodules per gram of fresh weight of root of Glycine max treated with a solvent containing a control peptide at 0.01 µM (left bar) or with a solvent containing 0.1 µM of miPEP 172c (right bar). The error bar corresponds to the standard error of the mean (number of individuals = 12). The presence of an asterisk indicates a significant difference in expression between the two conditions tested. FIGURE 6 . Effects of miPEP172c on miR172c expression and gene expression NNC1, NSP1, NIN, ENOD40-1, Hb2 And nitH at the house of Glycine max. The y-axis indicates the relative expression of miR172c and genes NNC1, NSP1, NIN, ENOD40-1, Hb2 and nifHin a control plant watered with a solvent containing a control peptide at 0.01 µM (white columns) or in a plant watered with a solvent containing miPEP172c at 0.01 µM (black columns). The error bar corresponds to the standard error of the mean (number of individuals = 6). The presence of an asterisk indicates a significant difference in expression between the two conditions tested. FIGURE 7 . Effects of miPEP172c on root mass in Glycine max. The y-axis indicates the fresh root weight in grams in a control plant watered with a solvent containing a control peptide at 0.01 µM (left column) or in a plant watered with a solvent containing miPEP172c at 0.01 µM (right column) (number of individuals = 6). FIGURE 8 . Gene expression NifD. The photographs represent the expression of nifD revealed by nifD-LacZ fusions in nodules treated with the control peptide (A) or with miPEP172c (B). FIGURE 9 . Effects of miPEP167c on nodule number in Glycine max The y-axis indicates the number of nodules per gram of fresh weight of root of Glycine max treated with a solvent (control, left bar) or with a solvent containing 0.1 µM miPEP167c (miPEP167c, right bar). The error bar corresponds to the standard error of the mean (number of individuals = 12). EXAMPLES

[0364] miPEP172c increases the expression of miR172c ( Figures 1 And 6 ). The effect of miPEP172c is agonistic to that of miR172c, reducing gene expression NNC1 (repressive transcription factor) and causing an increase in gene expression NSP1, NIN, ENOD40-1, Hb2 and NifH involved in nodulation ( Figure 6 ).

[0365] Watering soybean plants ( Glycine max ) by low concentrations of miPEP172c (0.1 µM) specific to miR172c significantly increases the number of nodules ( Figures 2 And 5 ) and the nitrogen content of the aerial parts ( Figure 3), as well as the fresh weight of the pods ( Figure 4 ). No effect is observed on root development ( Figure 7 ). An increase in the number of inactive nodules sometimes occurs as a compensatory mechanism in response to reduced nitrogen fixation. Analysis of gene expression NifH by RT-qPCR ( Figure 6 ) and the observation of mergers nifD::LacZ ( Figure 8 ) however indicate efficient nitrogen fixation in plants treated with miPEP172c.

[0366] Taken together, these results indicate that treatment with miPEP172c mimics the effects of miR172c overexpression, both at the molecular and phenotypic levels. Watering soybean plants ( Glycine max ) by low concentrations of miPEP167c (0.1 µM) specific for miR167c also significantly increases the number of nodules ( Figure 9 ). Materials and methods • Measurement of miR172c and NNC1, NSP1, NIN and ENOD40-1 gene expression in Glycine max

[0367] Total RNA from roots of soy was extracted using the RNeasy Plant Mini Kit (Qiagen). Reverse transcription was performed using SuperScript II Reverse Transcriptase (Invitrogen) from 500 ng of total RNA. Three replicates (n=3) were performed with two technical replicates each. Each experiment was repeated two to three times. qPCR amplifications were performed using a LightCycler 480 System thermocycler (Roche Diagnostics) according to the method described in Lauressergues et al. (Plant J., 72(3):512-22, 2012). ELF1b was used as a housekeeping gene to normalize qRT-PCR analyses. The primers used for gene amplification ELF1b, NNC1, NSP1, NIN, ENOD40-1, Hb2 And nifHare described in the articles by Wang et al. (Soybean miR172c targets the repressive AP2 transcription factor NNC1 to activate ENOD40 expression and regulate nodule initiation, Plant cell 26(12): 4782-4801, 2014; MicroRNA167-directed regulation of the auxin response factors GmARF8a and GmARF8b is required for soybean nodulation and lateral root development, Plant Physiol 168(3): 984-999, 2015). • Germination of G. max seeds:

[0368] After sterilizing the seeds for 3 minutes in bleach diluted to ¼, then rinsing with sterile water, the seeds are incubated for 2 hours in water. Finally, the seeds are placed in a petri dish with damp paper at 28°C. Once the seeds have germinated, they are placed in a small pot containing oil dry ®< medium under a bell jar. After a few days of growth, plants with uniform development are selected for the experiment and transferred to a larger pot. • Inoculation of germinated seedlings with Bradirhizobium japonicum:

[0369] The bacteria are cultured in Campbel liquid medium (K 2 PO 4 : 0.5g / l, MgSO 4 7H 2 O : 0.2g / l, NaCl : 0.1g / l, Mannitol: 10g / l, Yeast extract: 2.5g / l, Casamino acid: 0.5g / l) (5 ml) at 28°C, then transferred into an Erlenmeyer flask containing 100ml of Campbel medium. After a few days of culture, when the OD is 0.3 at 595 nm, the 100 ml of culture of B. japonicum were centrifuged for 30 min at 4000 rpm. The supernatant was discarded and the pellet resuspended in “caisson” culture medium (Lullien et al., Plant gene expression in effective and ineffective root nodules of alfalfa (Medicago sativa). Plant Mol Biol 9: 469-478, 1987) to reach an OD of 0.05 at 595 nm. Each plant was inoculated with 20 ml of this solution. The plants were regularly watered with “caisson” medium. • Plant treatments with miPEP:

[0370] Treatment is performed by watering the plants with the concentrated miPEP solution or the equivalent solvent solution for the control treatment. A control treatment is also performed with a solvent solution containing a control peptide (SEQ ID NO: 5, VLWCSHCMGFLWPTYT). The control peptide and miPEP172c have the same amino acid content, but different sequences. Treatments are performed every other day.

Claims

1. Use of a miPEP exogenously introduced into a plant for promoting nodulation, between said plant and a bacterium, said miPEP being also naturally present in said plant, said exogenously introduced miPEP being either a peptide produced outside the plant or a peptide produced in the plant following the non-natural introduction of a nucleic acid encoding said miPEP into said plant, said exogenously introduced miPEP being a peptide having an amino acid sequence comprising or consisting of a sequence identical to that of a miPEP naturally present in said plant, said miPEP naturally present in said plant being a peptide of from 3 to 100 amino acids the sequence of which is coded by an open reading frame at the 5' portion of a primary transcript of a miR, said miPEP being capable of modulating the accumulation of said miR in said plant, which miR regulates the expression of at least one gene involved in the nodulation in said plant, the sum of the amount of said exogenously introduced miPEP and of the amount of said naturally present miPEP being strictly greater than the amount of said naturally present miPEP, said miPEP being the miPEP172c having an amino acid sequence having at least 80% identity with the amino acid sequence SEQ ID NO: 2, in particular said miPEP172c having an amino acid sequence consisting of SEQ ID NO: 2, preferably, said miR being the miR172c having in particular a nucleotide sequence consisting of SEQ ID NO: 1.

2. Use according to claim 1, wherein said plant is: - a leguminous plant, such as lotus (Lotus sp.), soybean (Glycine max), peanut (Arachis hypogaea), common bean (Phaseolus vulgaris), pea (Pisum sativum), lentil (Lens culinaris), chickpea (Cicer arietinum), broad bean and field bean (Vicia faba), vetches (Vicia sp.), vetchlings (Lathyrus sp.), alfalfa (Medicago sp.), clover (Trifolium sp.), lupin (Lupinus sp.), mungo bean (Vigna radiata), liquorice (Glycyrrhiza glabra), rosewood (Dalbergia), trefoil (Lotus corniculatus), sainfoin (Onobrychis viciifolia), rooibos (Aspalathus linearis), or fenugreek (Trigonella foenum-graecum), or - a sugar beet (Beta vulgaris).

3. Use according to any one of the preceding claims, wherein said bacterium is a bacterium from the Rhizobiaceae family, in particular selected from the genera Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium or Azorhizobium.

4. Method for promoting the nodulation between a plant and a bacterium, comprising a step of introducing a miPEP exogenously into a plant, said miPEP being also present naturally in said plant, said exogenously introduced miPEP being a peptide having an amino acid sequence comprising or consisting of a sequence identical to that of a miPEP naturally present in said plant, said exogenously introduced miPEP being a peptide of from 3 to 100 amino acids the sequence of which comprises or consists of a sequence identical to that of said naturally present miPEP, which sequence of the naturally present miPEP is coded by an open reading frame at the 5' portion of the primary transcript of a miR, said miPEP being capable of modulating the accumulation of said miR, which miR regulates the expression of at least one gene involved in the nodulation in said plant, the sum of the amount of said exogenously introduced miPEP and of the amount of said naturally present miPEP being strictly greater than the amount of said naturally present miPEP, said miPEP being the miPEP172c having an amino acid sequence having at least 80% identity with the sequence SEQ ID NO: 2, in particular said miPEP172c having an amino acid sequence consisting of the sequence SEQ ID NO: 2, preferably, said miR being miR172c having in particular a nucleotide sequence consisting of SEQ ID NO: 1, said bacterium being in particular of the Rhizobiaceae family, said plant being in particular a leguminous plant.

5. Method according to claim 4, for promoting nodulation between a Glycine max plant and a Bradirhizobium japonicum bacterium, wherein miPEP172c is exogenously introduced into said Glycine max plant, said miPEP172c being naturally present in said Glycine max plant, said miPEP172c exogenously introduced being a peptide comprising or consisting of a sequence identical to that of said naturally present miPEP172c, which naturally present miPEP172c is a peptide of from 3 to 100 amino acids of which the sequence is coded by an open reading frame at the 5' portion of the primary transcript of the miR172c, said miPEP172c being capable of increasing the accumulation of said miR172c, which miR172c regulates the expression of at least one gene involved in the nodulation in Glycine max, the sum of the amount of said miPEP172c exogenously introduced and of the amount of said naturally present miPEP172c being strictly greater than the amount of said naturally present miPEP172c.

6. Method according to claim 4 or 5, wherein said miPEP is administered to the plant: - externally, preferably by watering, by spraying, or by the addition of a fertiliser, a soil, a culture substrate, or with the aid of a support in contact with the plant, said miPEP being in particular administered to the plant in the form of a composition comprising from 10-9 M to 10-4 M of said miPEP, especially 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 the addition of a fertiliser, a soil, a culture substrate, or with the aid of a support in contact with the grain or the seed, said miPEP being in particular administered to the grain or the seed in the form of a composition comprising from 10-9 M to 10-4 M of said miPEP, especially 10-9, 10-8, 10-7, 10-6, 10-5 or 10-4 M of said miPEP, or - by way of a nucleic acid coding said miPEP, said nucleic acid being introduced into the plant, but said nucleic acid not comprising the complete sequence of the corresponding miR.

7. Method for producing a transgenic plant, said method comprising: a) a step of introducing a nucleic acid coding a miPEP having from 3 to 100 amino acids into a plant, but said nucleic acid not comprising the complete sequence of the corresponding miR, or into at least a cell of said plant, in conditions allowing the expression of said miPEP, said miPEP also being present naturally in said plant, said naturally present miPEP being a peptide of which the sequence is coded by an open reading frame at the 5' portion of the primary transcript of a miR, said miPEP being capable of modulating the accumulation of said miR in the plant, which miR regulates the expression of at least one gene involved in the nodulation, and b) a step of culturing the plant, or at least a cell of said plant, obtained in step a) in conditions making it possible to obtain a transgenic plant, said miPEP being the miPEP172c having an amino acid sequence having at least 80% identity with the amino acid sequence SEQ ID NO: 2, in particular said miPEP172c having an amino acid sequence consisting of SEQ ID NO: 2, preferably, said miR being the miR172c having in particular a nucleotide sequence consisting of SEQ ID NO: 1.

8. Transgenic plant such as obtained by the method according to claim 7.

9. Peptide having an amino acid sequence having at least 80% identity, preferably at least 90% identity, with the amino acid sequence SEQ ID NO: 2, preferably said peptide comprising or consisting of sequence SEQ ID NO: 2, said peptide being capable of modulating the accumulation of miR172c.

10. Composition comprising: - miPEP172c as an active substance, said miPEP172c comprising or consisting of an amino acid sequence having at least 80% identity with the sequence SEQ ID NO: 2, in particular said miPEP172c comprising or consisting of an amino acid sequence consisting of SEQ ID NO: 2, said miPEP172c being in particular at a concentration from 10-9 M to 10-4 M of said miPEP, especially 10-9, 10-8, 10-7, 10-6, 10-5 or 10-4 M, or in particular, said composition further comprising an excipient, a diluent, or a solvent, in particular, said composition being formulated so as to form a coated product.

11. Composition comprising, in combination, a quantity of seeds of a plant and a quantity of a synthetic peptide of which the sequence comprises or consists of a sequence identical to that of a miPEP naturally present in said plant, said synthetic peptide having a sequence comprising or consisting of a sequence identical to that of miPEP172c, said miPEP172c having an amino acid sequence showing at least 80% identity with the sequence SEQ ID NO: 2, in particular said miPEP172c having an amino acid sequence consisting of SEQ ID NO: 2, in particular, said composition being formulated so as to form a coated seed.

12. Coated seed with a composition comprising a miPEP capable of modulating the accumulation of a miR in the plant produced from the seed, which miR regulates the expression of at least one gene involved in the nodulation in said plant, said miPEP being the miPEP172c having an amino acid sequence having at least 80% identity with the amino acid sequence SEQ ID NO: 2, in particular said miPEP172c having an amino acid sequence comprising or consisting of SEQ ID NO: 2.

13. Use of a miPEP for culturing bacteria, in particular said bacterium being of the Rhizobiaceae family, said use comprising a step of contacting said bacteria with: - a mixture comprising a plant or a plant part, especially a root culture, and a peptide of which the sequence comprises or consists of a sequence identical to that of a miPEP naturally present in said plant, said naturally present miPEP being a peptide, the sequence of which is coded by an open reading frame at the 5' portion of the primary transcript of a miR, said miPEP being capable of modulating the accumulation of said miR, which miR regulates the expression of at least one gene involved in the nodulation in said plant, or - a transgenic plant as defined above, the plant, the plant part, and the transgenic plant being able to form a symbiosis with said bacterium, said miPEP being the miPEP172c having an amino acid sequence having at least 80% identity with the sequence SEQ ID NO: 2, in particular said miPEP172c having an amino acid sequence consisting of the sequence SEQ ID NO: 2.

14. Use of a miPEP for producing a bacterial inoculum, in particular an inoculum of bacteria from the Rhizobiaceae family, said use comprising: - a step in which bacteria are co-cultured with a living plant material, referred to as the host plant, corresponding at least in part to a constituent part of the root of a plant capable of forming a nodulation with said bacteria, et - a step of contacting a quantity of a peptide with said co-culture, said peptide having a sequence comprising or consisting of a sequence identical to that of a miPEP naturally present in said host plant, said naturally present miPEP being a peptide, the sequence of which is coded by an open reading frame at the 5' portion of the primary transcript of a miR, said miPEP being capable of modulating the accumulation of said miR, which miR regulates the expression of at least one gene involved in the nodulation in said host plant, said miPEP being the miPEP172c having an amino acid sequence having at least 80% identity with the amino acid sequence SEQ ID NO: 2, in particular said miPEP172c having an amino acid sequence consisting of SEQ ID NO: 2.

15. Bacterial inoculum, in particular an inoculum of bacteria from the Rhizobiaceae family, suitable for the inoculation of a host plant, comprising at least one bacterium and a peptide, the sequence of which comprises or consists of a sequence identical to that of a miPEP naturally present in the host plant, said miPEP naturally present in the host plant being a peptide, the sequence of which is coded by an open reading frame at the 5' portion of the primary transcript of a miR, said miPEP being capable of modulating the accumulation of said miR, which miR regulates the expression of at least one gene involved in the nodulation in said host plant, said miPEP being the miPEP172c having an amino acid sequence having at least 80% identity with the amino acid sequence SEQ ID NO: 2, in particular said miPEP172c having an amino acid sequence consisting of SEQ ID NO: 2.