MicroRNAs (miRNAs) are small regulatory RNA molecules that inhibit the expression of specific target genes by binding to and cleaving their messenger RNAs or otherwise inhibiting their translation into proteins1. miRNAs are transcribed as much larger primary transcripts (pri-miRNAs), the function of which is not fully understood. Here we show that plant pri-miRNAs contain short open reading frame sequences that encode regulatory peptides. The pri-miR171b of Medicago truncatula and the pri-miR165a of Arabidopsis thaliana produce peptides, which we term miPEP171b and miPEP165a, respectively, that enhance the accumulation of their corresponding mature miRNAs, resulting in downregulation of target genes involved in root development. The mechanism of miRNA-encoded peptide (miPEP) action involves increasing transcription of the pri-miRNA. Five other pri-miRNAs of A. thaliana and M. truncatula encode active miPEPs, suggesting that miPEPs are widespread throughout the plant kingdom. Synthetic miPEP171b and miPEP165a peptides applied to plants specifically trigger the accumulation of miR171b and miR165a, leading to reduction of lateral root development and stimulation of main root growth, respectively, suggesting that miPEPs might have agronomical applications.
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This work was funded by the French ANR project miRcorrhiza (ANR-12-JSV7-0002-01), the CNRS, Paul Sabatier University Toulouse. This work is also supported by Toulouse Tech Transfer (http://www.toulouse-tech-transfer.com) for valorization and transfer. It was carried out in the LRSV which belongs to the Laboratoire d'Excellence entitled TULIP (ANR-10-LABX-41). We thank the GenoToul bioinformatics facility for providing computing and storage resources. We also thank J.-M. Prospéri (UMR AGAP 1334, Montpellier, France) for M. truncatula seeds, X. Chen (University of California, USA) for nrpb2-3 seeds, V. Cotelle (LRSV) for help with protein analyses, C. Rosenberg (LIPM, Castanet Tolosan, France) for providing modified pCAMBIA 2200, and F. Payre and S. Plaza (CBD CNRS, Toulouse), J. Cavaillé (LBME CNRS, Toulouse) and C. Featherstone for critical reading of the manuscript.
The authors declare no competing financial interests.
Extended data figures and tables
The 5′ part of the pri-miR171b, as identified by 5′ RACE–PCR analysis. The short putative ORFs are in blue, the miR171b
a–c, Staining for GUS activity (blue) showing expression of the miR171b at different stages of lateral root development. d–f, Staining for GUS activity (blue) showing expression of the miPEP171b at different stages of lateral root development. g–r, Immunolocalization of miPEP171b. Confocal immunofluorescence microscopy of endogenous miPEP171b (red) in the main roots (g) and lateral root primordia (i) of wild-type plants, and their corresponding bright-field images (h, j). Confocal immunofluorescence microscopy of miPEP171b (red) in main roots (k) and lateral root primordia (m) in roots overexpressing miPEP171b, and their corresponding bright-field images (l, n). Controls for immunofluorescence staining of main roots (o) and lateral root primordia (q) of wild-type plants, and their corresponding bright-field images (p, r), in which the primary antibody was omitted (scale bars, 100 μm). One representative experiment of nine (g–j), four (k–n) or seventeen (o–r) performed is shown.
Extended Data Figure 3 Effect of an upregulation of the miR171b on root development in M. truncatula.
a, Relative production of lateral roots in roots overexpressing the pri-miR171b compared to the production in control roots. b, Relative production of lateral roots in roots overexpressing the miPEP171b compared to the production in control roots. c, Relative production of lateral roots in plants treated with 0.1 μM miPEP171b synthetic peptide (0.1 µM miPEP171b) compared to the production in roots treated with control solvent (control). Error bars represent s.e.m., asterisks indicate a significant difference between the overexpressing roots and the control according to Student’s t-test (n = 100 independent plants, P < 0.05).
a, Effect of overexpression of miPEP171b. b, Effect of exogenous treatment with 0.1 µM of the synthetic miPEP171b. Histograms represent the relative expression of each miRNA in roots overexpressing miPEP171b compared to wild-type roots (a) or in roots treated with the peptide compared to the roots treated with solvent (b). Error bars represent s.e.m., asterisks indicate a significant difference between the treatment and the control according to a Kruskal–Wallis test (n = 10 independent roots, P < 0.05).
Extended Data Figure 5 Alignment of miPEP165a amino acid sequences of seven species of Brassicales using MultAlin.
MultAlin available at http://multalin.toulouse.inra.fr/multalin/multalin.html. At, Arabidopsis thaliana; Aly, Arabidopsis lyrata; Bn, Brassica napus; Bo, Brassica oleracea; Bc, Brassica carinata; Bj, Brassica juncea; Br, Brassica rapa. Red represents homology in all seven species; blue represents homology in most of the species. Upper-case letters denote high-consensus sequence; lower-case letters denote low-consensus sequence.
a, miPEP165a sequence: the 5′ part of the pri-miR165a, as identified in ref. 7. The miPEP165a-coding sequence is in blue, the miR165a* in green, and the mature miR165a in red. The putative alternative start codons are in light blue, the ATG start codon of the miPEP165a is underlined, and the amino acid sequence of the miPEP165a is shown below. The black vertical line indicates the 5′ end of the pre-miR165a precursor (http://www.mirbase.org). b–f, Expression of the miPEP165a in A. thaliana roots. b, c, Confocal fluorescence microscopy of A. thaliana root tips showing expression of a GUS-GFP fusion gene fused to the first ATG of the sequence encoding the pri-miR165a under the control of 4 kb of the promoter (green), and the corresponding bright-field image. The red fluorescence is due to the constitutively expressed DsRed fluorescent protein. d, A root tip as in (b, c) showing staining for GUS activity (blue) due to expression of the GUS-GFP fusion gene. e, f, Absence of staining for GUS activity in root tips transformed with expression vectors encoding GUS fusion genes fused to the first GTG (e) or CTG (f) of the pri-miR165a-coding sequence under the control of 4 kb of the promoter (scale bars, 100 μm). g, Main root length of seedlings treated with water (control) or with various concentrations of synthetic miPEP165a dissolved in water. Error bars represent s.e.m., asterisks indicate a significant difference between the treatment and the control according to Student’s t-test (n = 100 independent plants, P < 0.05).
Extended Data Figure 7 Effect of various miPEPs from Arabidopsis thaliana and Medicago truncatula on the expression of their corresponding miRNA.
a–c, miPEPs from A. thaliana. a, Expression of miR160b in response to overexpression of miPEP160b. b, Expression of miR164a in response to treatment with 0.1 µM of miPEP164a. c, Expression of miR319a in response to overexpression of miPEP319a. d–e, miPEPs from M. truncatula. d, Expression of miR169d in response to treatment with 0.1 µM of miPEP169d. e, Expression of miR171e in response to overexpression of miPEP171e. Error bars represent s.e.m., asterisks indicate a significant difference between the treatment and the control according to a Kruskal–Wallis test (n = 10 independent roots or tobacco leaves, P < 0.05).
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Lauressergues, D., Couzigou, JM., Clemente, H. et al. Primary transcripts of microRNAs encode regulatory peptides. Nature 520, 90–93 (2015). https://doi.org/10.1038/nature14346
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