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Negative feedback regulation of microbe-associated molecular pattern-induced cytosolic Ca2+ transients by protein phosphorylation

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Abstract

Microbe/pathogen-associated molecular patterns (MAMPs/PAMPs) often induce rises in cytosolic free Ca2+ concentration ([Ca2+]cyt) and protein phosphorylation. Though they are postulated to play pivotal roles in plant innate immunity, their molecular links and the regulatory mechanisms remain largely unknown. To investigate the regulatory mechanisms for MAMP-induced Ca2+ mobilization, we have established a transgenic rice (Oryza sativa) cell line stably expressing apoaequorin, and characterized the interrelationship among MAMP-induced changes in [Ca2+]cyt, production of reactive oxygen species (ROS) and protein phosphorylation. Oligosaccharide and sphingolipid MAMPs induced Ca2+ transients mainly due to plasma membrane Ca2+ influx, which were dramatically suppressed by a protein phosphatase inhibitor, calyculin A (CA). Hydrogen peroxide and hypo-osmotic shock triggered similar [Ca2+]cyt elevations, which were not affected by CA. MAMP-induced protein phosphorylation, which is promoted by CA, has been shown to be required for ROS production and MAPK activation, while it negatively regulates MAMPs-induced Ca2+ mobilization and may play a crucial role in temporal regulation of [Ca2+]cyt signature.

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Acknowledgments

We would like to thank Prof. Dr. Naoto Shibuya of Meiji University for gift of N-cetylchitooligosaccharides and suggestions, Prof. Dr. Kenzo Nakamura for the pIG121-Hm plasmid, Prof. Dr. Ko Shimamoto for suggestions, and Mr. Jumpei Hamada, Mr. Yohei Iwasaki, and Mr. Hiroshi Nokajima for technical assistance. This work was supported in part by Grant-in-Aid for Scientific Research on Innovative Areas (21200067 to T.K.), for Exploratory Research (21658118 to K.K.) and for Young Scientists (B) (21780041 to T.K.), and by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Integrated research project for plants, insects, and animals using genome technology MP-2134 to K.K.).

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Author notes

  1. Yasuhiro Kadota

    Present address: The Sainsbury Laboratory, Norwich Research Park, Norwich, NR4 7UH, UK

  2. Teruyuki Hayashi

    Present address: Division of Plant Sciences, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki, 305-8602, Japan

Authors and Affiliations

  1. Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510, Japan

    Takamitsu Kurusu, Haruyasu Hamada, Yoshimi Sugiyama, Toshikazu Yagala, Yasuhiro Kadota, Teruyuki Hayashi & Kazuyuki Kuchitsu

  2. Institute of Plant Science and Resources, Okayama University, Chuo 2-20-1, Kurashiki, Okayama, 710-0046, Japan

    Takuya Furuichi

  3. Agricultural and Veterinary Research Laboratories, Meiji Seika Kaisha Ltd, Yokohama, 222-8567, Japan

    Kenji Umemura

  4. National Institute of Crop Science, Kannondai 2-1-18, Tsukuba, 305-8518, Japan

    Setsuko Komatsu

  5. Division of Genome and Biodiversity Research, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki, 305-8602, Japan

    Akio Miyao & Hirohiko Hirochika

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  1. Takamitsu Kurusu
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Correspondence to Kazuyuki Kuchitsu.

Additional information

T. Kurusu, H. Hamada and Y. Sugiyama contributed equally to this work.

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Supplemental figure S1

RT-PCR analysis of the apoaequorin transformant cell lines. Rice calli (Oryza sativa cultivar Nipponbare) were transformed with apoaequorin gene targetted to the cytoplasm. The gene expression was monitored by RT-PCR analysis. First-strand cDNA was synthesized from total RNA extracted from each cell line and amplified by RT-PCR as described in Materials and methods. Ubiquitin cDNA was used as a control. PCR products were analyzed by agarose gel electrophoresis. (TIFF 814 kb)

Supplemental figure S2

The effect of several inhibitors on the N-acetylchitooligosaccharides-induced [Ca2+]cyt changes. Neomycin (500 μM), U-73122 (1 μM), and LaCl3 (5 mM) were added to the cells 15 min prior to the MAMP treatment (10 μM). Average values and SE of five independent experiments are shown. * P<0.05, ** P<0.01, significant difference as compared with the control according to unpaired Student’s t test. (TIFF 491 kb)

Supplemental figure S3

Ionomycin-induced changes in [Ca2+]cyt in rice (Oryza sativa) cells. Data are representatives of five experiments with similar results. rlu, relative luminescence unit. (TIFF 768 kb)

Supplemental figure S4

The Effect of a CaM antagonist, W7, on N-acetylchitooligosaccharides-induced [Ca2+]cyt changes. W7 (100 μM) were added to the rice (Oryza sativa) cells 15 min prior to the MAMP treatment (10 μM). Data are representatives of three experiments. rlu, relative luminescence unit. (TIFF 868 kb)

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Kurusu, T., Hamada, H., Sugiyama, Y. et al. Negative feedback regulation of microbe-associated molecular pattern-induced cytosolic Ca2+ transients by protein phosphorylation. J Plant Res 124, 415–424 (2011). https://doi.org/10.1007/s10265-010-0388-4

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