Defensin-like polypeptide LUREs are pollen tube attractants secreted from synergid cells

Abstract

For more than 140 years, pollen tube guidance in flowering plants has been thought to be mediated by chemoattractants derived from target ovules1. However, there has been no convincing evidence of any particular molecule being the true attractant that actually controls the navigation of pollen tubes towards ovules. Emerging data indicate that two synergid cells on the side of the egg cell emit a diffusible, species-specific signal to attract the pollen tube at the last step of pollen tube guidance1,2,3. Here we report that secreted, cysteine-rich polypeptides (CRPs) in a subgroup of defensin-like proteins are attractants derived from the synergid cells. We isolated synergid cells of Torenia fournieri, a unique plant with a protruding embryo sac, to identify transcripts encoding secreted proteins as candidate molecules for the chemoattractant(s). We found two CRPs, abundantly and predominantly expressed in the synergid cell, which are secreted to the surface of the egg apparatus. Moreover, they showed activity in vitro to attract competent pollen tubes of their own species and were named as LUREs. Injection of morpholino antisense oligomers against the LUREs impaired pollen tube attraction, supporting the finding that LUREs are the attractants derived from the synergid cells of T. fournieri.

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Figure 1: Identification and expression analysis of major CRPs of the synergid cell.
Figure 2: In vitro pollen tube attraction assay using recombinant CRPs.
Figure 3: Microinjection of MO antisense oligos for LUREs.

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Acknowledgements

We thank T. Suzuki for help with the analysis of the EST sequences, S. Takayama for providing chemically synthesized and refolded SP11 peptides, W. Uchida for checking the diameter of the glass needles for microinjection by scanning electron microscopy and N. Iwata for assistance in preparing plant materials. This work was supported in part by a Grant-in-Aid for Young Scientists (Start-up), Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan (20870020 to M.M.K.); a Grant-in-Aid for Creative Scientific Research, MEXT, Japan (18GS0314-01 to N.S.); a grant from Yamada Science Foundation, Japan (to T.H.); a Grant-in-Aid for Scientific Research (B), MEXT, Japan (19370017 to T.H.); a Grant-in-Aid for Scientific Research on Priority Areas (18075004 to T.H.), and PRESTO project, Japan Science and Technology Agency, Japan (to T.H.).

Author Contributions S.O., N.S. and T.H. developed methods to purify and assay recombinant LUREs. H.Tsutsui and T.H. developed methods for the microinjection of the MO oligos and performed the RT–PCR analysis. K.S., N.S. and M.M.K. developed immunological methods to detect LUREs. T.H., S.S. and T.D. constructed the cDNA library of the synergid cell of T. fournieri and directed the EST analysis. H.Takeuchi., R.Y., R.D.K., Y.H., A.M., D.S., N.K., T.S., K.I., K.O., M.M., H.N. and M.M.K. assembled and analysed the EST sequences. S.N. constructed expression vectors and purified recombinant LUREs. T.K., A.N., T.D., M.M.K., N.S. and T.H. contributed to the experimental design. T.H. directed the project and wrote the paper with input from co-authors.

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Correspondence to Tetsuya Higashiyama.

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Supplementary Information

This file contains Supplementary Figures 1-3 with Legends, Supplementary Tables 1-4 and the Legend for Supplementary Movie 1. (PDF 6881 kb)

Supplementary Movie 1

This movie file shows in vitro attraction assay using a pipette (see file s1 for full legend). (AVI 26776 kb)

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Okuda, S., Tsutsui, H., Shiina, K. et al. Defensin-like polypeptide LUREs are pollen tube attractants secreted from synergid cells. Nature 458, 357–361 (2009). https://doi.org/10.1038/nature07882

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