NOVEL METHOD FOR PROMOTING NODULATION IN PLANTS

Abstract

Micropeptides (miPEPs), or peptides coded by microRNAs, for promoting nodulation between a plant and a bacterium, as well as their use in this manner.

Claims

1-24. (canceled)

25. 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 miPEP introduced exogenously being a peptide comprising or consisting of a sequence identical to that of said naturally present miPEP, which naturally present miPEP 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 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 miPEP introduced exogenously and of the amount of said naturally present miPEP being strictly greater than the amount of said naturally present miPEP.

26. The method according to claim 25, wherein said miRNA is miR172c

27. The method according to claim 25, wherein said miRNA is miR172c and has a nucleotide sequence consisting of SEQ ID NO: 1.

28. The method according to claim 25, wherein said miPEP is miPEP172c.

29. The method according to claim 25, wherein said miPEP is miPEP172c and has an amino acid sequence consisting of SEQ ID NO: 2.

30. The method according to claim 25, wherein said miRNA is miR167c,

31. The method according to claim 25, wherein said miRNA is miR167c and has a nucleotide sequence consisting of SEQ ID NO: 6.

32. The method according to claim 25, wherein said miPEP is miPEP167c,

33. The method according to claim 25, wherein said miPEP is miPEP167c and has an amino acid sequence consisting of SEQ ID NO: 7.

34. The method according to claim 25, wherein said plant is: a leguminous plantselected from the group consisting of: 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), and fenugreek (Trigonella foenum-graecum), or sugar beet (Beta vulgaris).

35. The method according to claim 25, wherein said bacterium is a bacterium from the Rhizobiacea family selected from the genera Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium or Azorhizobium.

36. A method for producing a transgenic plant, comprising: a) a step of introducing a nucleic acid coding a miPEP having from 3 to 100 amino acids into a plant, 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.

37. The method for producing a transgenic plant according to claim 36 wherein said miR is miR172c, said miR172c having a nucleotide sequence consisting of SEQ ID NO: 1.

38. The method for producing a transgenic plant according to claim 36 wherein said miPEP is miPEP172c, said miPEP172c having an amino acid sequence consisting of SEQ ID NO: 2.

39. The method for producing a transgenic plant according to claim 36, wherein said miR is miR167c, said miR167c having a nucleotide sequence consisting of SEQ ID NO: 6.

40. The method for producing a transgenic plant according to claim 36, wherein said miPEP is miPEP167c, said miPEP167c having an amino acid sequence consisting of SEQ ID NO: 7.

41. A transgenic plant as obtained by the method as defined according to claim 36.

42. 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, said peptide comprising or consisting of SEQ ID NO: 2 or the amino acid sequence SEQ ID NO: 7, said peptide comprising or consisting of SEQ ID NO: 7.

43. A composition comprising a peptide according to claim 42 as active substance, said peptide being at a concentration of from 10.sup.9M to 10.sup.4M, said composition further comprising an excipient, a diluent, or a solvent, said composition being formulated so as to form a coated product.

44. A composition comprising, in combination, an amount of seeds of a plant and an amount of a peptide according to claim 42 of which the sequence comprises or consists of a sequence identical to that of a miPEP naturally present in said plant, said peptide having a sequence comprising or consisting of a sequence identical to that of the miPEP172c or of the miPEP167c, said composition being formulated so as to form a coated seed.

45. A bacterial inoculum of bacteria from the Rhizobiaceae family, suitable for inoculation of a host plant, comprising at least: one bacterium and one peptide of which the 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 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, which miR regulates the expression of at least one gene involved in the nodulation in said host plant.

Description

KEY TO THE DRAWINGS

[0348] FIG. 1. Effects of a treatment with miPEP172c on the expression of miR172c in Glycine max

[0349] The ordinate axis indicates the relative expression of miR172c in a control plant (left column) or in a plant watered with miPEP172c at 0.1 M (right column). The error bar corresponds to the standard error of the mean (number of individuals=9).

[0350] FIG. 2. Effects of miPEP172c on the number of nodules in Glycine max

[0351] The ordinate axis indicates the number of nodules per gram of fresh root weight of Glycine max treated with a solvent (control, left bar) or with a solvent containing 0.1 M of miPEP172c (miPEP172c, right bar).

[0352] The error bar corresponds to the standard error of the mean (number of individuals=9).

[0353] This experiment was repeated independently and gave similar results.

[0354] FIG. 3. Effects of miPEP172c on the nitrogen concentration in the aerial parts in Glycine max

[0355] The ordinate axis indicates the nitrogen concentration per dry leaf weight of Glycine max (in g/kg), treated with a solvent (control, left bar) or with a solvent containing 0.1 M of miPEP172c (miPEP172c, right bar).

[0356] The error bar corresponds to the standard error of the mean (number of individuals=9).

[0357] FIG. 4. Effects of miPEP172c on the fresh pod weight in Glycine max

[0358] The ordinate axis indicates the fresh dry pod weight in Glycine max (in g), treated with a solvent (control, left bar) or with a solvent containing 0.1 M of miPEP172c (miPEP172c, right bar).

[0359] The error bar corresponds to the standard error of the mean (number of individuals=9).

[0360] FIG. 5. Effects of miPEP172c on the number of nodules in Glycine max

[0361] The ordinate axis indicates the number of nodules per gram of fresh root weight of Glycine max treated with a solvent containing a control peptide at 0.01 M (left bar) or with a solvent containing 0.1 M of miPEP172c (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 of expression between the two conditions tested.

[0362] FIG. 6. Effects of miPEP172c on the expression of miR172c and on the expression of the genes NNC1, NSP1, NEV, ENOD40-1, Hb2 and nifH in Glycine max.

[0363] The ordinate axis indicates the relative expression of miR172c and of the genes NNC1, NSP1, NIN, ENOD40-1, Hb2 and nifH in a control plant watered with a solvent containing a control peptide at 0.01 M (white columns) or in a plant watered with a solvent containing miPEP172c at 0,01 M (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 of expression between the two conditions tested.

[0364] FIG. 7. Effects of miPEP172c on the root mass in Glycine max.

[0365] The ordinate axis indicates the fresh root weight in grams in a control plant watered with a solvent containing a control peptide at 0.01 M (left column) or in a plant watered with a solvent containing miPEP172c at 0.01 M (right column) (number of individuals=6).

[0366] FIG. 8. Expression of the gene NifD.

[0367] The photographs show the expression of nifD revealed by nifD-LacZ fusions in nodules treated with the control peptide (A) or with miPEP172c (B).

[0368] FIG. 9. Effects of miPEP167c on the number of nodules in Glycine max

[0369] The ordinate axis indicates the number of nodules per gram of fresh root weight of Glycine max treated with a solvent (control, left bar) or with a solvent containing 0.1 M of miPEP167c (miPEP167c, right bar).

[0370] The error bar corresponds to the standard error of the mean (number of individuals=12).

EXAMPLES

[0371] The miPEP172c increases the expression of miR172c (FIGS. 1 and 6). The effect of miPEP172c is agonistic to that of miR172c, reducing the expression of the gene NNC1 (repressive transcription factor) and prompting an increase in the expression of the genes NSP1, NIN, ENOD40-1, Hb2 and NifH involved in the nodulation (FIG. 6).

[0372] The watering of soybean plants (Glycine max) with low concentrations of miPEP172c (0.1 M) specific to miR172c significantly increases the number of nodules (FIGS. 2 and 5) and the nitrogen content of the aerial parts (FIG. 3), as well as the fresh pod weight (FIG. 4). No effect on the development of the roots was observed (FIG. 7).

[0373] A rise in the number of inactive nodules sometimes occurs as a compensation mechanism in response to a reduced fixation of nitrogen. The analysis of the expression of the gene NifH by RT-qPCR (FIG. 6) and the observation of nifD::LacZ fusions (FIG. 8), however, indicate effective fixation of nitrogen in the plants treated with miPEP172c.

[0374] All of these results indicate that the treatment with miPEP172c mimics the effects of an overexpression of miR172c, both at molecular and phenotype level.

[0375] The watering of soybean plants (Glycine max) with low concentrations of miPEP167c (0.1 M) specific to miR167c also significantly increases the number of nodules (FIG. 9).

Material and Methods

[0376] Measurement of the Expression of miR172c and of the Genes NNC1, NSP1, NIN and ENOD40-1 in Glycine max

[0377] The total RNA of the roots of soybean was extracted using the RNeasy Plant Mini Extraction Kit (Qiagen). The reverse transcription was performed using the reverse transcriptase SuperScript II (Invitrogen) from 500 ng of total RNA. Three repetitions (n=3) were performed with two technical repetitions each. Each experiment was repeated from two to three times. The amplifications by qPCR were performed using a LightCycler 480 Thermocycler System (Roche Diagnostics) in accordance with the method described in Lauressergues et al. (Plant J., 72(3):512-22, 2012). ELF1b was used as housekeeping gene to standardise the qRT-PCR analyses. The primers used for the amplification of the genes ELF1b, NNC1, NSP1, NIN, ENOD40-1, Hb2 and nifH are described in the articles of 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 grains:

[0378] After sterilisation of the grains for 3 min in bleach diluted , then rinsing with sterile water, the grains are incubated for 2 hours in water. Lastly, the grains are placed in a petri dish with damp paper towels at 28 C. Once sprouted, the grains are placed in a small pot containing oil dry medium under a glass cover. After a few days' growth, the plants of which the development is uniform are selected for the experiment and transferred into a larger pot.

Inoculation of the Sprouted Seedlings With Bradirhizobium japonicum:

[0379] The bacteria are placed in culture in Campbel liquid medium (K.sub.2PO.sub.4: 0.5g/l, MgSO.sub.4 7H.sub.2O: 0.2 g/l, NaCl: 0.1 g/l, mannitol: 10 g/l, yeast extract: 2.5 g/l, Casamino acid: 0.5 g/l) (5 ml) at 28 C., then transferred to an Erlenmeyer flask containing 100 ml of Campbel medium. After a few days of culture, when the DO 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 achieve a DO of 0.05 at 595 nm. Each plant is inoculated with 20 ml of this solution. The plants are regularly watered with the caisson medium.

Plant Treatments With the miPEPs:

[0380] The treatment is performed by watering plants with the concentrated solution of miPEP or the equivalent of 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 the miPEP172c have the same content of amino acids, but different sequences. The treatments are performed every two days.