Apoplastic plant subtilases support arbuscular mycorrhiza development in Lotus japonicus

Authors

  • Naoya Takeda,

    1. Faculty of Biology, Genetics, University of Munich, Großhaderner Straße 2, 82152 Martinsried, Germany
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    • Present addresses: NT; National Institute of Agrobiological Science, 2-1-2 Kan-non-dai, Tsukuba, Ibaraki 305-8602, Japan. EA; Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Ten-noudai, Tsukuba, Ibaraki 305-8572, Japan.

  • Shusei Sato,

    1. Kazusa DNA Research Institute, 2-6-7 Kazusa-kamatari, Kisarazu, Chiba 292-0818, Japan
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  • Erika Asamizu,

    1. Kazusa DNA Research Institute, 2-6-7 Kazusa-kamatari, Kisarazu, Chiba 292-0818, Japan
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    • Present addresses: NT; National Institute of Agrobiological Science, 2-1-2 Kan-non-dai, Tsukuba, Ibaraki 305-8602, Japan. EA; Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Ten-noudai, Tsukuba, Ibaraki 305-8572, Japan.

  • Satoshi Tabata,

    1. Kazusa DNA Research Institute, 2-6-7 Kazusa-kamatari, Kisarazu, Chiba 292-0818, Japan
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  • Martin Parniske

    Corresponding author
    1. Faculty of Biology, Genetics, University of Munich, Großhaderner Straße 2, 82152 Martinsried, Germany
      *(fax +49 2180 74702; e-mail parniske@lmu.de).
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*(fax +49 2180 74702; e-mail parniske@lmu.de).

Summary

In the arbuscular mycorrhiza (AM) symbiosis, plant roots accommodate Glomeromycota fungi within an intracellular compartment, the arbuscule. At this symbiotic interface, fungal hyphae are surrounded by a plant membrane, which creates an apoplastic compartment, the periarbuscular space (PAS) between fungal and plant cell. Despite the importance of the PAS for symbiotic signal and metabolite exchange, only few of its components have been identified. Here we show that two apoplastic plant proteases of the subtilase family are required for AM development. SbtM1 is the founder member of a family of arbuscular mycorrhiza-induced subtilase genes that occur in at least two clusters in the genome of the legume Lotus japonicus. A detailed expression analysis by RT-PCR revealed that SbtM1, SbtM3, SbtM4 and the more distantly related SbtS are all rapidly induced during development of arbuscular mycorrhiza, but only SbtS and SbtM4 are also up-regulated during root nodule symbiosis. Promoter–reporter fusions indicated specific activation in cells that are adjacent to intra-radical fungal hyphae or in cells that harbour them. Venus fluorescent protein was observed in the apoplast and the PAS when expressed from a fusion construct with the SbtM1 signal peptide or the full-length subtilase. Suppression of SbtM1 or SbtM3 by RNAi caused a decrease in intra-radical hyphae and arbuscule colonization, but had no effect on nodule formation. Our data indicate a role for these subtilases during the fungal infection process in particular arbuscule development.

Ancillary