Use of micropeptides in order to stimulate mycorrhizal symbiosis

Abstract

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

Claims

1. A method for promoting mycorrhizal symbiosis between a plant and a fungus, comprising a step of exogenously introducing a miPEP into the plant, said miPEP being naturally present in said plant, wherein the miPEP (i) is a peptide selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, and SEQ ID NO: 14, (ii) increases the accumulation of an miRNA in said plant relative to a plant that does not comprise exogenously introduced miPEP, and (iii) comprises or consists of an amino acid sequence of the natural miPEP encoded by the primary transcript of said miRNA; and wherein said miRNA is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, and SEQ ID NO: 13.

2. The method according to claim 1, wherein said miPEP is capable of modulating the accumulation of said miRNA in said plant, which miRNA regulates the expression of at least one gene involved in the mycorrhizal symbiosis in said plant, said gene is involved in the mycorrhizal symbiosis encoding a transcription factor of the GRAS family, said gene is involved in the mycorrhizal symbiosis being selected from the group consisting of: HAM1 and HAM2.

3. The method according to claim 1, wherein the mycorrhizal symbiosis is arbuscular mycorrhizal symbiosis.

4. The method according to claim 1, wherein the plant is a monocotyledon plant, a dicotyledon plant, a solanaceous plant, or a leguminous plant.

5. The method according to claim 1, wherein the plant is selected from the group consisting of: Medicago truncatula, Medicago sativa (alfalfa), Solanum lycopersicum (tomato), Lotus japonicus (birdsfoot trefoil), and Oryza sativa (rice).

6. The method according to claim 1, wherein the fungus is a glomeromycete, basidiomycete, or ascomycete.

7. The method according to claim 1, wherein the plant is a Medicago truncatula plant, the fungus is a glomeromycete fungus, and the miPEP is miPEP171b (SEQ ID NO: 2).

8. The method according to claim 1, wherein the plant is a Solanum lycopersicum plant, the fungus is a glomeromycete fungus, and the miPEP is slmiPEP171e (SEQ ID NO: 6).

9. The method according to claim 1, wherein the plant is a Lotus japonicus plant, the fungus is a glomeromycete fungus, and the miPEP is ljmiPEP171b (SEQ ID NO: 10).

10. The method according to claim 1, wherein the plant is an Oryza sativa plant, the fungus is a glomeromycete fungus, and the miPEP is osmiPEP171i (SEQ ID NO: 14).

11. The method according to claim 1, wherein: (a) the miRNA comprises the nucleotide sequence of SEQ ID NO: 1, and the miPEP comprises the amino acid sequence of SEQ ID NO: 2; (b) the miRNA comprises the nucleotide sequence of SEQ ID NO: 5, and the miPEP comprises the amino acid sequence of SEQ ID NO: 6; (c) the miRNA comprises the nucleotide sequence of SEQ ID NO: 9, and the miPEP comprises the amino acid sequence SEQ ID NO: 10; or (d) the miRNA comprises the nucleotide sequence of SEQ ID NO: 13, and the miPEP comprises the amino acid sequence of SEQ ID NO: 14, wherein the fungus is a glomeromycete and the plant is a monocotyledon plant, a dicotyledon plant, a solanaceous plant or a leguminous plant.

12. The method according to claim 1, wherein the exogenous introduction comprises: (a) administering to the plant of a composition comprising the miPEP at a concentration of 10.sup.9 M to 10.sup.4 M; (b) administering to a grain or a seed of a composition comprising the miPEP at a concentration of 10.sup.9 M to 10.sup.4 M, and cultivating the grain or the seed to produce the plant; or (c) administering to the plant a composition comprising a nucleic acid encoding the miPEP.

13. The method according to claim 12, wherein the administration comprises watering the plant with the composition, spraying the composition on the plant, or by adding a fertilizer comprising the composition to the plant.

14. The method according to claim 12, therein the concentration is selected from the group consisting of 10.sup.9, 10.sup.8, 10.sup.7, 10.sup.6, 10.sup.5, and 10.sup.4 M.

15. A method for the production of a transgenic plant comprising: a) introducing a nucleic acid encoding a miPEP into a plant, or at least one cell of the plant, under conditions allowing the expression of the miPEP, and b) growing the plant, or the at least one cell of said plant, obtained in step a) under conditions allowing a transgenic plant to be obtained, wherein the miPEP (i) is a peptide selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 6, SEQ ID NO:10 and SEQ ID NO:14, (ii) increases the accumulation of an miRNA in said plant relative to a plant that does not comprise exogenously introduced miPEP, and (iii) comprises or consists of an amino acid sequence of the natural miPEP encoded by the primary transcript of said miRNA; and wherein said miRNA is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, and SEQ ID NO: 13 and the nucleic acid consists of the sequence coding for said miRNA.

16. A transgenic plant as obtained by method of claim 15.

Description

CAPTIONS TO THE FIGURES

(1) FIG. 1. Effects of the overexpression of miR171b (miR171b identified in Medicago truncatula) on the expression of the HAM1 and HAM2 (A) genes or on the number of lateral roots (B) in M. truncatula.

(2) (A) The y-axis shows the relative expression of miR171b (left columns), of HAM1 (centre columns) or of HAM2 (right columns) in a control plant (white columns) or in a plant in which miR171b is overexpressed (black columns). The error bar corresponds to the standard error of the mean (number of individuals=10). The overexpression of miR171b causes a reduction in the expression of the HAM1 and HAM2 genes.

(3) (B) The y-axis shows the average number of lateral roots observed in a control plant (white column) or in a plant in which miR171b is overexpressed (black column). The error bar corresponds to the standard error of the mean (number of individuals=100). The overexpression of miR171b leads to a reduction in the number of lateral roots.

(4) FIG. 2. Effects of the overexpression of miPEP171b on the expression of miR171b and of the HAM1 and HAM2 (A) genes or on the number of lateral roots (B) in M. truncatula.

(5) (A) The y-axis shows the relative expression of miPEP171b (graph on the left), of miR171b (graph on the right, left columns), of HAM1 (accession No. MtGI9-TC114268) (graph on the right, centre columns) or of HAM2 (accession No. MtGI9-TC120850) (graph on the right, right columns) in a control plant (white columns) or in a plant in which miPEP171b is overexpressed (black columns). The error bar corresponds to the standard error of the mean (number of individuals=10). The overexpression of miPEP171b causes an increase in the accumulation of miR171b, as well as a reduction in the expression of the genes HAM1 and HAM2.

(6) (B) The y-axis shows the average number of lateral roots observed in a control plant (white column) or in a plant in which miPEP171b is overexpressed (black column). The error bar corresponds to the standard error of the mean (number of individuals=100). The overexpression of miPEP171b leads to a reduction in the number of lateral roots.

(7) FIG. 3. Effects of miPEP171b on the expression of miR171b and of the genes HAM1 and HAM2 (A) and on the number lateral roots (B) in M. truncatula.

(8) (A) The y-axis shows the relative expression of miR171b (left columns), of HAM1 (centre columns) or of HAM2 (right columns) in a control plant (white columns) or in a plant grown on a medium containing miPEP171b at 0.01 M (light grey columns), 0.1 M (dark grey columns) or 1 M (black columns). The error bar corresponds to the standard error of the mean (number of individuals=10). The application of miPEP171b to the different concentrations causes an increase in the accumulation of miR171b, as well as a reduction in the expression of the HAM1 and HAM2 genes.

(9) (B) The y-axis shows the average number of lateral roots observed in a control plant (white column) or in a plant grown on medium containing miPEP171b at 0.1 M for 5 days and 1 time per day (black column). The error bar corresponds to the standard error of the mean (number of individuals=100). The application of miPEP171b at 0.1 M leads to a reduction in the number of lateral roots.

(10) (C) The y-axis shows the relative expression of MtmiR171b (left columns), of HAM1 (centre columns) or of HAM2 (right columns) in a control plant (white columns) or in a plant treated by watering for 5 days and 1 time per day with MtmiPEP171b1 at 0.01 M (grey columns), 0.1 M (dark grey columns) or 1 M (black columns) or with 0.01 M of a peptide mixture (light grey columns) the composition of amino acids of which is identical to miPEP171b but the sequence of which is different. The error bar corresponds to the standard error of the mean (number of individuals=10).

(11) FIG. 4. Immunolocalization

(12) The roots of Medicago truncatula were transformed in order to express fusions between the protein GUS (in blue) and the ATG of miPEP171b (Pro.sub.miR171b-ATG1:GUS) or the ATG2 (second ATG being located on the precursor, after the miPEP) (Pro.sub.miR171b-ATG2:GUS). Labelling was also carried out with an antibody anti-miPEP171b (miPEP171b). The immunolocalization of miPEP171b in the roots of M. truncatula reveals the presence of miPEP171b in the initiation sites of the lateral roots, showing a co-localization between the microRNA and the corresponding miPEP.

(13) FIG. 5. Effects of miPEP171b on the colonization of M. truncatula by the fungus Rhizophagus irregularis

(14) The y-axis shows the percentage colonization (on the left) and the abundance of the arbuscules (on the right) in roots of M. truncatula treated with a solvent (control, light bars) or with a solvent containing 0.1 M of miPEP171b (miPEP171b, dark bars). The error bar corresponds to the standard error of the mean (number of individuals=15).

(15) FIG. 6. Effects of miPEP171b on the surface area of the arbuscules formed by the fungus Rhizophagus irregularis in M. truncatula

(16) The y-axis shows the surface area of the arbuscules (measured in arbitrary units) in roots of M. truncatula treated with a solvent (control) or with a solvent containing 0.1 M of miPEP171b (miPEP171b).

(17) The error bar corresponds to the standard error of the mean (number of individuals=15).

(18) FIG. 7. Effect of miPEP171b on the mycorrhization rate of M. truncatula by the fungus Rhizophagus irregularis

(19) The y-axis shows the mycorrhization rate in roots of M. truncatula treated 5 weeks post inoculation with a solvent (left bar) or with a solvent containing 0.1 M of miPEP171b (miPEP, right bar).

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

(21) FIG. 8. Effect of slmiPEP171e on the mycorrhization rate of Solanum lycopersicum

(22) The y-axis shows the mycorrhization rate of Solanum lycopersicum plants treated 12 weeks post inoculation with a solvent (left bar) or with a solvent containing 0.1 M of slmiPEP171e (miPEP, right bar).

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

(24) FIG. 9. Effects of ljmiPEP171b on the number of fungal structures in Lotus japonicus

(25) The y-axis shows the number of fungal structures in a plant treated 5 weeks post inoculation with a solvent (left bar) or with a solvent containing 0.1 M of ljmiPEP171b (Pep 171, right bar).

(26) The error bar corresponds to the standard error of the mean (number of individuals=10 control plants, 12 treated plants).

(27) FIG. 10. Effects of ljmiPEP171b on the number of intraradical hyphae in Lotus japonicus

(28) The y-axis shows the number of intraradical hyphae in a plant treated 5 weeks post inoculation with a solvent (left bar) or with a solvent containing 0.1 M of ljmiPEP171b (Pep 171, right bar).

(29) The error bar corresponds to the standard error of the mean (number of individuals=10 control plants, 12 treated plants).

(30) FIG. 11. Effects of ljmiPEP171b on the number of arbuscules in Lotus japonicus

(31) The y-axis shows the number of arbuscules in a plant treated 5 weeks post inoculation with a solvent (left bar) or with a solvent containing 0.1 M of ljmiPEP171b (Pep 171, right bar).

(32) The error bar corresponds to the standard error of the mean (number of individuals=10 control plants, 12 treated plants).

(33) FIG. 12. Effects of the ljmiPEP171b on the number of vesicles in Lotus japonicus

(34) The y-axis shows the number of vesicles in a plant treated 5 weeks post inoculation with a solvent (left bar) or with a solvent containing 0.1 M of ljmiPEP171b (Pep 171, right bar). The error bar corresponds to the standard error of the mean (number of individuals=10 control plants, 12 treated plants).

(35) FIG. 13. Effects of the osmiPEP171i on the number of fungal structures in Oryza sativa

(36) The y-axis shows the number of fungal structures in a plant treated 5 weeks post inoculation with a solvent (left bar) or with a solvent containing 0.1 M of ljmiPEP171b (Pep 171, right bar).

(37) The error bar corresponds to the standard error of the mean (number of individuals=5 control plants, 5 treated plants).

(38) FIG. 14. Effects of osmiPEP171i on the number of intraradical hyphae in Oryza sativa

(39) The y-axis shows the number of intraradical hyphae in a plant treated 5 weeks post inoculation with a solvent (left bar) or with a solvent containing 0.1 M of ljmiPEP171b (Pep 171, right bar).

(40) The error bar correspond to the standard error of the mean (number of individuals=5 control plants, 5 treated plants).

(41) FIG. 15. Effects of osmiPEP171i on the number of arbuscules in Oryza sativa

(42) The y-axis shows the number of arbuscules in a plant treated 5 weeks post inoculation with a solvent (left bar) or with a solvent containing 0.1 M of ljmiPEP171b (Pep 171, right bar).

(43) The error bar corresponds to the standard error of the mean (number of individuals=5 control plants, 5 treated plants).

(44) FIG. 16. Effects of osmiPEP171i on the number of vesicles in Oryza sativa

(45) The y-axis shows the number of vesicles in a plant treated 5 weeks post inoculation with a solvent (left bar) or with a solvent containing 0.1 M of OsmiPEP171i (Pep 171, right bar).

(46) The error bar corresponds to the standard error of the mean (number of individuals=5 control plants, 5 treated plants).

(47) FIG. 17. Effects of slmiPEP171e on the mycorrhization rate of Solanum lycopersicum

(48) The y-axis shows the mycorrhization rate of Solanum lycopersicum plants treated 12 weeks post inoculation with a solvent (left bar) or with a solvent containing 0.1 M of slmiPEP171e (right bar).

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

(50) FIG. 18. Effects of ljPEP171b on the mycorrhization rate of Lotus japonicus

(51) The y-axis shows the mycorrhization rate of Lotus japonicus plants treated 5 weeks post inoculation with a solvent (left bar) or with a solvent containing 0.1 M of ljmiPEP171b (right bar).

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

(53) FIG. 19. Effects of OsPEP171i on the mycorrhization rate of Oricea sativa

(54) The y-axis shows the mycorrhization rate of Oryza sativa plants treated 5 weeks post inoculation with a solvent (left bar) or with a solvent containing 0.1 M of OsmiPEP171i (right bar).

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

EXAMPLES

Example 1Identification and Characterization of miPEP171b

(56) MiR171b is expressed in the meristematic region of the roots, as well as in the initiation sites of the lateral roots. The overexpression of this miRNA leads in particular to a reduction in the expression of the HAM1 genes (Accession No. MtGI9-TC114268) and HAM2 genes (Accession No. MtGI9-TC120850) (FIG. 1A), as well as a reduction in the number of lateral roots (FIG. 1B).

(57) The sequence of the primary transcript of the miR171b was determined by using the RACE-PCR technique. Analysis of the sequence of the primary transcript made it possible to identify the presence of several small open reading frames (ORF) that were completely unexpected.

(58) The overexpression of the first ORF, called miORF171b, leads to an increase in the accumulation of miR171b and a reduction in the expression of the HAM1 and HAM2 genes (see FIG. 2A), as well as to a reduction in the number of lateral roots (FIG. 2B), as was already observed during the overexpression of miR171b.

(59) In order to determine if miORF171b leads to the actual production of a peptide an immunolocalization of the peptide with a specific antibody was carried out and revealed the presence of the peptide in the initiation sites of the lateral roots, thus showing a co-localization between the microRNA and the corresponding miPEP. In order to determine if the regulation function noted is in fact shown by said peptide, a synthetic peptide, the sequence of which is identical to that potentially encoded by miORF171b, was applied to the roots of Medicago truncatula. The application of this peptide leads to the phenotype already noted above during the overexpression of miORF171b, i.e. it leads to an increase in the accumulation of miR171b and a reduction in the expression of the HAM1 and HAM2 genes (see FIG. 3A), as well as a reduction in the number of lateral roots (FIG. 3B). The results of these experiments demonstrate that the miORF171b encoded a peptide capable of modulating the accumulation of miR171b, and the expression of the target genes miR171b: HAM1 and HAM2. Said peptide was called miPEP171b.

(60) Moreover, the immunolocalization of miPEP171b1 in the roots of M. truncatula reveals the presence of miPEP171b1 in the initiation sites of the lateral roots, showing a co-localization between the microRNA and the corresponding miPEP.

Example 2Effects of miPEP171b on Mycorrhization in M. truncatula

(61) Medicago truncatula plants were treated for 5 weeks, by watering every 2 days, with low concentrations (0.1 M) of a synthetic peptide the sequence of which is identical to that of miPEP171b, and the percentage colonization of the plant was measured.

(62) The results of these experiments indicate that the treatment with the miPEP171b significantly increased the mycorrhization in M. truncatula (FIG. 5).

(63) Moreover, it was observed that the arbuscules obtained in the roots treated with miPEP171b have a larger size than those present in the control roots not treated with miPEP171b (FIG. 6).

(64) It was also observed that the mycorrhization rate is greater in the plants treated with miPEP171b relative to the control plants not treated with miPEP171b (FIG. 7).

Example 3Effects of miPEP171b on Mycorrhization in Medicago sativa

(65) The homologue of the miPEP171b of M. truncatula was identified in M. sativa by BLAST and RACE PCR.

(66) A treatment with different doses of miPEP171b (0.01 M to 10 M) is carried out in parallel with the mycorrhization of M. sativa.

Example 4Effects of miPEP171b on Mycorrhization in Glycine max (Soya)

(67) The homologue of the miPEP171b of M. truncatula was identified in soya by BLAST and RACE PCR.

(68) A treatment with different doses of miPEP171b (0.01 M to 10 M) is carried out in parallel with mycorrhization of the soya.

Example 5Effects of slmiPEP171e on Mycorrhization in Solanum lycopersicum (Tomato)

(69) The homologue of the miPEP171e of M. truncatula was identified in the tomato by BLAST and RACE PCR.

(70) The effects of slmiPEP171e on mycorrhization of the tomato were analyzed by treating the plants with slmiPEP171e in parallel with the mycorrhization.

(71) The results are shown in FIGS. 8 and 17.

(72) These results indicate that slmiPEP171e promotes the mycorrhization of the tomato.

Example 6Effects of LjmiPEP171b on Mycorrhization in Lotus japonicus (Birdsfoot Trefoil)

(73) The homologue of the miPEP171b of M. truncatula was identified in birdsfoot trefoil by BLAST and RACE PCR.

(74) The effects of the LjmiPEP171b on mycorrhization of the birdsfoot trefoil were analyzed by treating the plants with LjmiPEP171b in parallel with the mycorrhization.

(75) The results are shown in FIGS. 9, 10, 11, 12 and 18.

(76) These results indicate that LjmiPEP171b promotes the mycorrhization of birdsfoot trefoil.

Example 7Effects of osmiPEP171i on Mycorrhization in Oryza sativa (Rice)

(77) The homologue of the miPEP1 71i of M truncatula was identified in rice by BLAST and RACEPCR.

(78) The effects of the osmiPEP171i on mycorrhization of the rice were analyzed by treating the plants with osmiPEP171i in parallel with the mycorrhization.

(79) The results are shown in FIGS. 13, 14, 15, 16 and 19. These results indicate that osmiPEP171i promotes the mycorrhization of birdsfoot trefoil rice.

(80) Material and Methods

(81) Medicago truncatula

(82) Biological Material

(83) The surface of the seeds of M. truncatula was sterilized and they were placed to germinate on agar plates for 5 days at 4 C. in the dark. The young shoots were then grown on 12 cm square plates filled with Fahraeus medium without nitrogen and containing 7.5 M phosphate (Lauresergues et al., Plant J., 72(3):512-22, 2012). The lateral roots were counted every day. In pots, the plants were watered every two days with modified Long Ashton medium containing little phosphorus (Balzergue et al., 2011 Journal of experimental botany, 62:1049-1060).

(84) The peptides were synthesized by Eurogentec or Smartox-Biotech. The miPEP171b was placed in suspension in a water 40%/acetonitrile 50%/acetic acid 10% (v/v/v) solution.

(85) Reverse Transcription of the microRNAs

(86) The RNA was extracted by using the reagent Tri-Reagent (MRC) according to the manufacturer's instructions, with the exception of precipitation of the RNA which was carried out with 3 volumes of ethanol. The reverse transcription of the RNA was carried out by using the specific stem-loop primer RTprimer171b in combination with hexamers for carrying out the reverse transcription of the RNA of high molecular weight.

(87) In brief, 1 g of RNA was added to the stem-loop primer MIR171b (0.2 M), the hexamer (500 ng), the buffer RT (1), the SuperScript Reverse transcriptase (SSIII) enzyme (one unit), the dNTPs (0.2 mM each), the DTT (0.8 mM) in a final reaction mixture of 25 l. In order to carry out the reverse transcription, a pulsed reverse transcription reaction was carried out (40 repetitions of the following cycle: 16 C. for 2 minutes, 42 C. for one minute and 50 C. for one second, followed by a final inactivation of the reverse transcription at 85 C. for 5 minutes).

(88) Analyses by Quantitative RT-PCR (qRT-PCR)

(89) The total RNA of the roots of M. truncatula was extracted by using the RNeasy Plant Mini Kit extraction kit (Qiagen). The reverse transcription was carried out by using the reverse transcriptase SuperScript II (Invitrogen) starting from 500 ng of total RNA. Three repetitions (n=3) were carried out, each with two technical repetitions. Each experiment was repeated from two to three times. The amplifications by qPCR were carried out by using a LightCycler 480 System (Roche Diagnostics) thermocycler according to the method described in Lauressergues et al. (Plant J., 72(3):512-22, 2012).

(90) Statistical Analyses

(91) The mean values of the relative expression of the genes or of the production of lateral roots were analyzed by using the Student test or the Kruskal-Wallis test. The error bars represent the standard error of the mean (SEM). The asterisks indicate a significant difference (p<0.05).

(92) Plasmid Constructions

(93) The DNA fragments of interest were amplified with Pfu polymerase (Promega). The DNA fragments were cloned using the XhoI and NotI enzymes in a pPEX-DsRED plasmid for an overexpression under the control of the strong constitutive promoter 35S, and using the KpnI-NcoI enzymes in a pPEX GUS plasmid for the reporter genes, according to the method described in Combier et al. (Genes & Dev, 22: 1549-1559, 2008).

(94) Transformation of the Plants

(95) The composite plants having roots transformed with Agrobacterium Rhizogenes were obtained by the method described in Boisson-Dernier et al. (Mol Plant-Microbe Interact, 18:1269-1276, 2005). The transformed roots were verified and selected by observations of DsRED with a binocular fluorescence magnifier. The control roots correspond to roots transformed with A. rhizogenes not containing the pPEX-DsRED vector.

(96) Mycorrhization

(97) Sterile spores of Rhizophagus irregularis (previously called Glomus intraradices) DAOM197198 were purchased from Agronutrition (Carbonne, France).

(98) Seeds of M. truncatula (Gaertn Jemalong genotype A17) were sterilized on the surface and placed to germinate on agar plates in the dark for 5 days at 4 C. The plants were then grown on an Oil-Dri US-special substrate (Damolin, Denmark) for 5 to 12 weeks in a growing room and watered every 2 days with modified Long Ashton medium containing a low concentration of phosphate (Balzergue et al., J Exp Bot, 62(3):1049-60, 2011).

(99) For the inoculation of the plants with R. irregularis, 450 spores were used per plant.

(100) After washing in KOH 10% (weight/volume) and rinsing in sterile water, the mycorrhized roots were treated for 30 minutes with wheat germ agglutinin fluorescein conjugate (Invitrogen) which fixes the fungal chitin, then washed 3 times in a PBS buffer. The roots were then observed with an inverted optical microscope or a confocal microscope (Leica, France).

(101) Alternatively, the roots were labelled with black Schaeffer ink according to the protocol described in Vierheilig et al., Appl Environ Microbiol, 64:5004-5007, 1998.

(102) The percentage mycorrhization was measured using grids according to the protocol described in Giovannetti and Mosse, New Phytol, 84:489-500, 1980.

(103) The accurate phenotyping of the mycorrhization was also carried out according to the method of Trouvelot et al., Physiological and Genetical Aspects of Mycorrhizae, pp 217-221, 1986. The frequency of mycorrhization (F) in the root system and the abundance of the arbuscules (A) (as a percentage) were calculated in colonized root sections using the software Mycocalc.

(104) The size of the arbuscules was measured using the software ImageJ.

(105) Each mycorrhization experiment was carried out at least twice by using 12 plants for each condition, each corresponding to an independent conversion with A. rhizogenes.

(106) Immunolocalization

(107) Roots or seedlings of tissues of Medicago were fixed for 2 hours in 4% formol (v/v) with 50 mM of phosphate buffer (pH 7.2), then included in LMP agarose 5% in water (with a low melting point). Thin sections (100 m) were obtained and placed in Pbi (phosphate buffer for immunology) on Teflon-coated slides, blocked in Pbi, 2% Tween and 1% bovine serum albumin for 2 hours (PbiT-BSA), then marked overnight (12 h) at 4 C. with the primary antibody diluted in BSA-PbiT. The sections were washed with PBiT and incubated at ambient temperature for 2 h with a secondary antibody diluted in PbiT-BSA. The slides were then washed in Pbi for 30 min and mounted in citifluor (mounting medium). The primary antibody and the dilutions were as follows: 1716a (1:500, v/v). The secondary antibody was a goat anti-rabbit IgG antibody coupled with the Alexa Fluorine 633 fluorescent probe (Molecular Probes), and was used at a dilution of 1:1000 (v/v).

(108) S. lycopersicum

(109) Germination of the Seeds of S. lycopersicum

(110) The seeds are sterilized for 2 minutes in sodium hypochlorite solution diluted , then rinsed at least 8 times with sterile water.

(111) The seeds are placed in a Petri dish containing water+1% agar. The dishes are placed at 4 C. overnight, then they are placed at ambient temperature for 4 days in order to germinate the seeds.

(112) Inoculation of the Germinated Seedlings:

(113) The germinated seeds are transferred to an Oil-Dry substrate containing spores of Rhizophagus irregularis at the concentration of 400 spores/L. After two days of adaptation under mini-greenhouse the seedlings are transferred to a growing room or greenhouse. The plants are watered regularly with a solution of Long Ashton+nitrogen (Lauressergues et al., Plant J. 72(3):512-22, 2012).

(114) Treatments of the Plants with miPEP:

(115) The treatment is carried out by watering the plants with 12.5 ml of water+the concentrated solution of miPEP or the equivalent of solvent solution (50% acetonitrile) for the control treatment. The treatments are carried out every two days.

(116) Tomato Root Staining Protocol:

(117) The roots are cleared with 1% KOH 7 minutes at 90 C., then stained 10 minutes in an ink solution (5% Sheaffer ink; 5% acetic acid) at 90 C.

(118) L. japonicus

(119) Germination of the Seeds of L. japonicus:

(120) The seeds are sterilized in sodium hypochlorite solution for 20 min then rinsed several times in water.

(121) Inoculation of the Germinated Seedlings:

(122) Culturing of the birdsfoot trefoil plants is carried out in a pot containing vermiculite with cotton fibre at the bottom. 5,000 spores of Rhizophagus irregularis are added per pot. The spores in mixtures in a substrate are arranged in a layer on the vermiculite to approximately two thirds of the height of the pot. The remainder of the pot is then filled with vermiculite and 10 seeds of pre-germinated birdsfoot trefoil are placed in the moist substrate. 3 pots are prepared for each treatment. The pots are then places in a growing room at 25 C.

(123) Treatments of the Plants with miPEP: LjmiPEP171e: 0.1 M (final) Control: water.

(124) The treatments are carried out on Monday and Friday by mixing the solution of miPEP or control with the nutrient solution (suitable B&D medium) and on Wednesday the solution of miPEP or control in water. Each pot is watered with 10 ml except on Friday 30 ml (without changing the quantity of miPEP), in order to last over the weekend.

(125) Birdsfoot Trefoil Staining Protocol:

(126) Protocol adapted from Vierheilg et al. (1998) (Ink vinegar a simple staining technique for arbuscular-mycorrhizal fungi. Environ. Microbiol. 64 (12): 5004-7) Cut the roots of the plants inoculated with the mycorrhizal fungus, wash and place them in a 2 ml tube. Cover the samples with a solution of 10% KOH and clear the tissues for 15 min at 95 C. in a heating unit. Wear gloves when handling the hot KOH. Drain the KOH and rinse the roots once with water and once with 10% acetic acid. Care must be taken not to touch the roots as they are very fragile. Cover the roots with a solution of black ink (Pelikan) 5% in a solution of 5% acetic acid. Heat for 5 minutes at 95 C. Carefully remove the ink solution with a 200 L pipette without damaging the roots. Rinse the roots twice with water. Clear the roots in a 5% acetic acid solution for 20 minutes under stirring. Store at 4 C. until use. Mount the roots between slide and cover slip in water or in 50% glycerol.

(127) O. sativa

(128) Germination of the Seeds of O. sativa:

(129) The seeds are sterilized for 1 min with 70% ethanol, then for 30 min with sodium hypochlorite solution before rinsing.

(130) Inoculation of the Germinated Seedlings:

(131) Culturing of a previously germinated seed of rice is carried out in a pot containing vermiculite with cotton fibre at the bottom. 500 spores of Rhizophagus irregularis are added per pot. The spores in mixtures in a substrate are arranged in a layer on the vermiculite at approximately two thirds of the height of the pot. The remainder of the pot is then filled with vermiculite and a hole is made for placing a germinated rice seed therein. The substrate is moistened. The pots are then placed in a growing room at 25 C.

(132) Treatments of the Plants with miPEP: OsmiPEP171i: 0.1 M (final) Control: equivalent of 50% ACN solution

(133) The treatments are carried out on Monday and Friday by mixing the solution of miPEP or control in 60 ml of nutrient solution (1/2 Hoagland with 25 M Pi and Sequestren) and on Wednesday the solution of miPEP or control in 60 ml of water. Each plant is watered with 10 ml per pot.

(134) Rice Root Staining Protocol:

(135) The roots can be stored in 10% KOH for several months before being stained. Incubation of the roots for 30 minutes at 96 C. in KOH in a 2 ml tube (with gentle stirring). Remove the KOH Rinse three times with water Incubate in 0.3M HCl for 15 min to 2 h. Remove the HCl Add approximately 1 ml of 0.1% Trypan blue and incubate the samples for 5 min at 96 C. Remove the Trypan blue Wash the samples in 50% acidic glycerol and mount the root sections between slide and cover slip.