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Kinase activity-dependent stability of calcium/calmodulin-dependent protein kinase of Lotus japonicus

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Abstract

Main conclusion

Accumulation of calcium/calmodulin-dependent protein kinase (CCaMK) in root cell nucleus depends on its kinase activity but not on nuclear symbiotic components crucial for nodulation.

Abstract

Plant calcium/calmodulin-dependent protein kinase (CCaMK) is a key regulator of symbioses with rhizobia and arbuscular mycorrhizal fungi as it decodes symbiotic calcium signals induced by microsymbionts. CCaMK is expressed mainly in root cells and localizes to the nucleus, where microsymbiont-triggered calcium oscillations occur. The molecular mechanisms that control CCaMK localization are unknown. Here, we analyzed the expression and subcellular localization of mutated CCaMK in the roots of Lotus japonicus and found a clear relation between CCaMK kinase activity and its stability. Kinase-defective CCaMK variants showed lower protein levels than the variants with kinase activity. The levels of transcripts driven by the CaMV 35S promoter were similar among the variants, indicating that stability of CCaMK is regulated post-translationally. We also demonstrated that CCaMK localized to the root cell nucleus in several symbiotic mutants, including cyclops, an interaction partner and phosphorylation target of CCaMK. Our results suggest that kinase activity of CCaMK is required not only for the activation of downstream symbiotic components but also for its stability in root cells.

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Abbreviations

CaMBD:

CaM-binding domain

CCaMK:

Ca2+/calmodulin (CaM)-dependent protein kinase

RFP:

Red fluorescent protein

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Acknowledgements

This work was financially supported by the National Agriculture and Food Research Organization of Japan.

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Authors and Affiliations

  1. Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan

    Yoshikazu Shimoda, Haruko Imaizumi-Anraku & Makoto Hayashi

  2. Plant Symbiosis Research Team, RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa, Japan

    Makoto Hayashi

Authors
  1. Yoshikazu Shimoda
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  2. Haruko Imaizumi-Anraku
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  3. Makoto Hayashi
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Correspondence to Yoshikazu Shimoda.

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425_2019_3264_MOESM1_ESM.pdf

Supplementary material 1 Subcellular localization of kinase-defective and EF-hand deletion CCaMKs. a Images of CCaMK localization taken under longer exposure time than that in Figs. 1 and 3. Merge, merged images of RFP fluorescence and bright field. Scale bars = 50 μm. b Ratio of signal intensity in the nucleus and cytosol in WT, kinase-defective, and EF-hand deletion CCaMKs. Signal intensity was measured under the short (black bar) and long exposure time (white bar) because the signal intensity of WT CCaMK in the nucleus was saturated under the long exposure. The inset is an image of signal intensity measurement by ZEN software. Circular and square dashed lines represent the central area of the nucleus and cytosol where the signal intensity was measured, respectively. Bars represent the means with standard error. Different letters indicate significant difference (Tukey–Kramer multiple comparison test, P < 0.05). n indicates the number of cells analyzed (PDF 651 kb)

425_2019_3264_MOESM2_ESM.pdf

Supplementary material 2 Nuclear localization of CCaMK in the symbiotic mutants and its interaction with CYCLOPS. a Nuclear localization of red fluorescent protein (RFP)-fused wild-type CCaMK in the roots of symbiotic mutants. Merge, merged images of RFP fluorescence and bright field. Nuclear localization of wild-type CCaMK was observed in all analyzed symbiotic mutants. Scale bars = 50 μm. b Yeast two-hybrid analysis showing the interaction between CCaMK and CYCLOPS. CYCLOPS Q107stop and W371stop are the truncated CYCLOPS which correspond the cyclops-4 and cyclops-3 mutations, respectively. Yeast growths on the selection mediums (SD-LWH and SD-ALWH) are shown. Growth on non-selective medium (SD-LW) is shown for successful yeast mating of bait and prey clones. pAS2-1 and pACT2 are the empty vectors for bait and prey clones, respectively (PDF 367 kb)

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Shimoda, Y., Imaizumi-Anraku, H. & Hayashi, M. Kinase activity-dependent stability of calcium/calmodulin-dependent protein kinase of Lotus japonicus. Planta 250, 1773–1779 (2019). https://doi.org/10.1007/s00425-019-03264-6

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