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Quantifying the plant actin cytoskeleton response to applied pressure using nanoindentation

  • New Methods in Cell Biology
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Abstract

Detection of potentially pathogenic microbes through recognition by plants and animals of both physical and chemical signals associated with the pathogens is vital for host well-being. Signal perception leads to the induction of a variety of responses that augment pre-existing, constitutive defences. The plant cell wall is a highly effective preformed barrier which becomes locally reinforced at the infection site through delivery of new wall material by the actin cytoskeleton. Although mechanical stimulation can produce a reaction, there is little understanding of the nature of physical factors capable of triggering plant defence. Neither the magnitude of forces nor the contact time required has been quantified. In the study reported here, mechanical stimulation with a tungsten microneedle has been used to quantify the response of Arabidopsis plants expressing an actin-binding protein tagged with green fluorescent protein (GFP) to reveal the organisation of the actin cytoskeleton. Using confocal microscopy, the response time for actin reorganisation in epidermal cells of Arabidopsis hypocotyls was shown to be 116 ± 49 s. Using nanoindentation and a diamond spherical tip indenter, the magnitude of the forces capable of triggering an actin response has been quantified. We show that Arabidopsis hypocotyl cells can detect a force as small as 4 μN applied for as short a time as 21.6 s to trigger reorganisation of the actin cytoskeleton. This force is an order of magnitude less than the potential invasive force determined for a range of fungal and oomycete plant pathogens. To our knowledge, this is the first quantification of the magnitude and duration of mechanical forces capable of stimulating a structural defence response in a plant cell.

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    Authors and Affiliations

    1. Plant Science Division, Research School of Biology, College of Medicine, Biology and Environment, The Australian National University, Canberra, ACT, Australia

      Rémi Branco, Chelsea A. Rundle & Adrienne R. Hardham

    2. Electronic Materials Engineering, Research School of Physics and Engineering, College of Physical and Mathematical Sciences, The Australian National University, Canberra, ACT, Australia

      Eliza-Jane Pearsall & Jodie E. Bradby

    3. CSIRO Agriculture, Canberra, ACT, Australia

      Rosemary G. White

    Authors
    1. Rémi Branco
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    2. Eliza-Jane Pearsall
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    3. Chelsea A. Rundle
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    4. Rosemary G. White
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    5. Jodie E. Bradby
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    6. Adrienne R. Hardham
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    Correspondence to Adrienne R. Hardham.

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    Handling Editor: Peter Nick

    Rémi Branco and Eliza-Jane Pearsall contributed equally to this work.

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    Supplementary Fig. 1

    Actin microfilament aggregation beneath the second site of contact with a tungsten microneedle in an Arabidopsis hypocotyl epidermal cell. The first point of contact resulted in the formation of the actin patch on the left (black arrow). The microneedle was then moved to a second position on the right (white arrowhead). The seven images are maximum projections of seven optical sections taken 56–104 s after the second contact was initiated. In this experiment, the first indication of actin aggregation (arrows) at the second point of stimulation was observed 80 s after probe contact. Scale bar = 20 μm. (GIF 382 kb)

    High resolution image (TIF 2188 kb)

    Supplementary Fig. 2

    The width of Arabidopsis hypocotyl epidermal cells as a function of plant age. Cell widths were measured from maximal projections used for actin response time determinations. Error bars show standard deviation. (GIF 5 kb)

    High resolution image (TIF 61 kb)

    Supplementary Fig. 3

    Diagram showing indenter probe positioning in the centre (green) or flanks (red) of epidermal cells. Contact on the flanks of the cell can lead to slippage of the probe (blue arrows) towards the groove overlying the anticlinal cell wall between adjacent epidermal cells. (GIF 11 kb)

    High resolution image (TIF 1396 kb)

    Supplementary Table 1

    Response time (in s) for actin aggregation in Arabidopsis hypocotyl epidermal cells after microneedle contact under different conditions (DOCX 14 kb)

    Supplementary Table 2

    Actin aggregation responses observed with different indenter tip geometries. (DOCX 12 kb)

    Supplementary Table 3

    Summary of measurements of turgor pressure and invasive forces generated by fungal and oomycete plant pathogens reported in the literature. (DOCX 28 kb)

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    Branco, R., Pearsall, EJ., Rundle, C.A. et al. Quantifying the plant actin cytoskeleton response to applied pressure using nanoindentation. Protoplasma 254, 1127–1137 (2017). https://doi.org/10.1007/s00709-016-0984-9

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    • DOI: https://doi.org/10.1007/s00709-016-0984-9

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