Whole-mount confocal imaging of nuclei in giant feeding cells induced by root-knot nematodes in Arabidopsis

Authors

  • Paulo Vieira,

    1. Institut National de la Recherche Agronomique, UMR 1355 ISA, 400 route des Chappes, Sophia-Antipolis, France
    2. Centre National de la Recherche Scientifique, UMR 7254 ISA, 400 route des Chappes, Sophia-Antipolis, France
    3. Université de Nice-Sophia Antipolis, UMR ISA, 400 route des Chappes, Sophia-Antipolis, France
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  • Gilbert Engler,

    1. Institut National de la Recherche Agronomique, UMR 1355 ISA, 400 route des Chappes, Sophia-Antipolis, France
    2. Centre National de la Recherche Scientifique, UMR 7254 ISA, 400 route des Chappes, Sophia-Antipolis, France
    3. Université de Nice-Sophia Antipolis, UMR ISA, 400 route des Chappes, Sophia-Antipolis, France
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    • These authors contributed equally to this work.

  • Janice de Almeida Engler

    1. Institut National de la Recherche Agronomique, UMR 1355 ISA, 400 route des Chappes, Sophia-Antipolis, France
    2. Centre National de la Recherche Scientifique, UMR 7254 ISA, 400 route des Chappes, Sophia-Antipolis, France
    3. Université de Nice-Sophia Antipolis, UMR ISA, 400 route des Chappes, Sophia-Antipolis, France
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    • These authors contributed equally to this work.


Author for correspondence:
Janice de Almeida Engler
Tel: +33 492 386 459
Email: janice.almeida-engler@sophia.inra.fr

Summary

  • Excellent visualization of nuclei was obtained here using a whole-mount procedure adapted to provide high-resolution images of large, irregularly shaped nuclei. The procedure is based on tissue clearing, and fluorescent staining of nuclear DNA with the dye propidium iodide.
  • The method developed for standard confocal imaging was applied to large multicellular root swellings, named galls, induced in plant hosts by the root-knot nematode Meloidogyne incognita.
  • Here, we performed a functional analysis, and examined the nuclear structure in giant feeding cells overexpressing the cell cycle inhibitor Kip-related protein 4 (KRP4). Ectopic KRP4 expression in galls led to aberrant nuclear structure, disturbing giant cell expansion and nematode reproduction. In vivo live-cell imaging of GFP-KRP4 demonstrated that this protein co-localizes to chromosomes from prophase to late anaphase during cell cycle progression.
  • The data presented here suggest the involvement of KRP4 during mitotic progression in plant cells. The detailed results obtained using confocal analysis also demonstrate the potential utility of a rapid, easy-to-use clearing method for the analysis of the nuclei of certain Arabidopsis mutants and other complex plant nuclei.

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