Control of female gamete formation by a small RNA pathway in Arabidopsis

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Abstract

In the ovules of most sexual flowering plants female gametogenesis is initiated from a single surviving gametic cell, the functional megaspore, formed after meiosis of the somatically derived megaspore mother cell (MMC)1,2. Because some mutants and certain sexual species exhibit more than one MMC2,3,4, and many others are able to form gametes without meiosis (by apomixis)5, it has been suggested that somatic cells in the ovule are competent to respond to a local signal likely to have an important function in determination6. Here we show that the Arabidopsis protein ARGONAUTE 9 (AGO9) controls female gamete formation by restricting the specification of gametophyte precursors in a dosage-dependent, non-cell-autonomous manner. Mutations in AGO9 lead to the differentiation of multiple gametic cells that are able to initiate gametogenesis. The AGO9 protein is not expressed in the gamete lineage; instead, it is expressed in cytoplasmic foci of somatic companion cells. Mutations in SUPPRESSOR OF GENE SILENCING 3 and RNA-DEPENDENT RNA POLYMERASE 6 exhibit an identical defect to ago9 mutants, indicating that the movement of small RNA (sRNAs) silencing out of somatic companion cells is necessary for controlling the specification of gametic cells. AGO9 preferentially interacts with 24-nucleotide sRNAs derived from transposable elements (TEs), and its activity is necessary to silence TEs in female gametes and their accessory cells. Our results show that AGO9-dependent sRNA silencing is crucial to specify cell fate in the Arabidopsis ovule, and that epigenetic reprogramming in companion cells is necessary for sRNA–dependent silencing in plant gametes.

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Figure 1: Phenotypes of ago9 insertional mutants before meiosis.
Figure 2: Meiotic and post-meiotic phenotype of the ago9 mutant.
Figure 3: AGO9 protein expression in developing ovules.
Figure 4: Phenotype of the rdr6 mutant and activation of transposable elements in the ago9 mutant.

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EMBL/GenBank/DDBJ

Change history

  • 25 March 2010

    In the sentence beginning, ‘In all ago9-3 ovules observed…’ a typographical error was corrected on 25 March 2010.

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Acknowledgements

We thank N. Sánchez for sharing the pFM2 marker, E. Demunck for technical assistance during cloning and sequencing, S. Poethig, J. Carrington, T. Lagrange and the Arabidopsis Stock Center for providing mutants, J. Mendiola and C. Alvarez for help with genetic and bioinformatic analysis, and R. Jorgensen for critically reading the manuscript. This work was supported by IRD-France and ANR (D.A. and D.G.), NIH and NSF (R.K.S. and R.A.M), Consejo Nacional de Ciencia y Tecnología (V.O.-M., N.D.-F., M.A.-V., E.D.-A. and J.-P.V.-C.), Consejo Estatal de Ciencia y Tecnología de Guanajuato (J.-P.V.-C.), and the Howard Hughes Medical Institute (J.-P.V.-C.).

Author Contributions J.-P.V.-C. and V.O.-M. designed the research, V.O.-M. generated the phenotypic analysis, performed the histological and expression analysis, and conducted the genetic experiments, N.D.-F. designed the antibody and performed the immunoprecipitations and sRNA analysis, M.A.-V. conducted the bioinformatic expression analysis, E.D.-A. performed immunolocalization experiments, D.G. contributed ideas and performed immunolocalization experiments, R.K.S. and D.A. provided unpublished materials, R.A.M. contributed ideas and J.-P.V.-C. wrote the paper.

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Correspondence to Jean-Philippe Vielle-Calzada.

Additional information

sRNA sequences are deposited in the EMBL Nucleotide Sequence Database (FN649764 to FN650107).

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This file contains Supplementary Figures 1-7 with legends, Supplementary Tables 1-2, and Supplementary References. (PDF 1398 kb)

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Olmedo-Monfil, V., Durán-Figueroa, N., Arteaga-Vázquez, M. et al. Control of female gamete formation by a small RNA pathway in Arabidopsis. Nature 464, 628–632 (2010) doi:10.1038/nature08828

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