Perception of soft mechanical stress in Arabidopsis leaves activates disease resistance
© Benikhlef et al.; licensee BioMed Central Ltd. 2013
Received: 15 May 2013
Accepted: 1 August 2013
Published: 13 September 2013
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© Benikhlef et al.; licensee BioMed Central Ltd. 2013
Received: 15 May 2013
Accepted: 1 August 2013
Published: 13 September 2013
In a previous study we have shown that wounding of Arabidopsis thaliana leaves induces a strong and transient immunity to Botrytis cinerea, the causal agent of grey mould. Reactive oxygen species (ROS) are formed within minutes after wounding and are required for wound–induced resistance to B. cinerea.
In this study, we have further explored ROS and resistance to B. cinerea in leaves of A. thaliana exposed to a soft form of mechanical stimulation without overt tissue damage. After gentle mechanical sweeping of leaf surfaces, a strong resistance to B. cinerea was observed. This was preceded by a rapid change in calcium concentration and a release of ROS, accompanied by changes in cuticle permeability, induction of the expression of genes typically associated with mechanical stress and release of biologically active diffusates from the surface. This reaction to soft mechanical stress (SMS) was fully independent of jasmonate (JA signaling). In addition, leaves exposed soft mechanical stress released a biologically active product capable of inducing resistance to B. cinerea in wild type control leaves.
Arabidopsis can detect and convert gentle forms of mechanical stimulation into a strong activation of defense against the virulent fungus B. cinerea.
Plants are exposed to various forms of mechanical stress caused by rain, snow, wind, animals, pathogens or plants themselves. Such mechanical stimuli induce responses in the plant that were shown in many cases to have an adaptive value . A classical example is the response of trees to wind that results in shorter and thicker trunks. Reaction or compression wood is an anatomical consequence of sensing mechanical stress with subsequent lignification of cell walls [2, 3]. Plants also respond to a more delicate mechanical stress referred to as touch that leads to nastic or tropic responses (thigmonasty or thigmotropism). Classical examples include the folding of Mimosa pudica’s leaflets, the leaf closure of the Venus fly trap or the coiling of tendrils . Such stimuli lead to visible responses such as a reorientation of organs or changes in shape allowing catching an insect or improved anchorage. The response of plants to mechanical stimuli can also be more discrete without any apparent overt changes. For example, mechanical stress associated with damage or wounds can lead to increased resistance to insects [5, 6] or fungal pathogens [7–9].
A closer look at the response to wounding has shown that it induces biochemical and molecular changes often associated with subsequently induced resistance mechanisms. For instance, wounded plants produce reactive oxygen species (ROS) [10, 11], undergo changes in lignification , JA, other hormones or wound signals  and exhibit changes in gene expression [5, 14] that are associated with induced defense reactions.
In a previous study we have shown that wounding of Arabidopsis thaliana leaves induces a strong and transient immunity to Botrytis cinerea the causal agent of grey mould . The expression of genes for camalexin biosynthesis and of glutathione-S-transferase, the activity of a MAP kinase activity and the accumulation of camalexin are primed by wounding . Wound-induced immunity is independent of the major plant defense pathways involving salicylic acid, JA or ethylene, but depends on glutathione . Recently, we have shown that wounding leads to the formation of ROS within minutes and ROS are required for wound–induced resistance to B. cinerea. A strong constitutive resistance to B. cinerea also takes place in mutants such as bdg and lacs2.3 defective in the production of a functional cuticle and displaying a phenotype of enhanced cuticular permeability . Moreover, leaf surfaces treated with cutinase produced ROS and became more protected to B. cinerea. Thus, increased permeability of the cuticle is linked to ROS formation and resistance to B. cinerea. In this study, we have further explored the responses of A. thaliana such as ROS and resistance to B. cinerea in leaves that are subjected to more gentle form of mechanical stimulation.
Wounding inflicted by clamping leaves with forceps or puncturing with a needle induces a strong immunity of A. thaliana to B. cinerea. In this study, we have explored the effect of softer forms of mechanical stimulation on the resistance of A. thaliana to B. cinerea. In particular, we have observed that a gentle mechanical stimulus applied to the surface of the leaf induced a transient and localized resistance to B. cinerea. Plants are known to be equipped with a sensitive and discriminative sensory system for the detection of molecular patterns generated by pathogens (pathogen- or microbe-associated molecular patterns, PAMPs or MAMPs; damage-associated molecular patterns, DAMPs) . Here we show that a gentle mechanical stress can also be perceived in a differentiated way and lead to specific plant responses that include resistance against a virulent necrotrophic fungus.
How does SMS compare to wounding? Overall, the results on SMS-induced resistance to B. cinerea overlap with wound-induced resistance of A. thaliana leaves . The results presented here further the published observations on wounding by showing that a soft mechanical friction of the surface layer without wounding the underlying cell is already enough to induce resistance. The absence of overt cellular breakage after SMS is supported by the absence of change in the levels of ABA after SMS under dry or humid conditions (Additional file 3) and the observation of leaf surfaces of WT and glabrous mutants after SMS (Figures 4 and 5A). Despite this, SMS-treated plants as well as the waxless mutant display an increased permeability to toluidine blue or Calcofluor white, indicating that the cuticular barrier is affected to a certain extent. The altered cuticular permeability might allow the diffusion of a bioactive molecule(s) observed in SMS-treated plants (Figure 9), similarly as in cuticle-defective mutants . Both wounding and SMS lead to a rapid and important release of ROS. A number of reports have associated mechanical stress with an increased production of ROS [22–25]. For example, rubbing tomato plant internodes results in a rapid and lasting accumulation of H2O2. ROS are well known for their effect as intracellular signals and were shown to be involved in the activation of defenses in response to biotic and abiotic stress . SMS-induced resistance to B. cinerea still takes place in atrbohD, atrbohF and atrbohD/F mutants of NADPH oxidoreductase making it unlikely that these NADPH oxidoreductases are involved (Additional file 1). It cannot be excluded that some other Arabidopsis NADPH oxidoreductases are involved. This is similar to results obtained with these mutants after wounding . In addition, SMS- like wound-induced resistance are both independent of JA signaling (Figure 3) .
How is SMS perceived by the plant? The results presented here lend themselves to a similar interpretation as the studies on resistance to B. cinerea observed in A. thaliana after wounding or in plants with defective cuticles [15, 20]. SMS might modify the plant surface making it more permeable thus allowing a better transfer of DAMPs (possibly produced by the disturbance of the surface upon SMS) or MAMPs (from B. cinerea) through the cell wall into the cell where they are recognized by adequate receptors. The changes in cuticular permeability (Figure 6), the ROS production (Figure 1) and the diffusion of bioactive molecule(s) through the surface (Figure 9) are all hallmarks of such a scenario. The slightly increased permeability of myb96-1 shown in 2 permeability tests (toluidine blue and Calcofluor white, Figure 8D-E) seems to be sufficient to allow for faster ROS production after inoculation and an increased resistance but not enough to allow sufficient leakage of active diffusates. Recent observations have shown a strong link between the presence of ROS and resistance to B. cinerea after wounding, and the data presented here agree with these conclusions. In fact, ROS accumulation and resistance after wounding were shown to depend on calcium changes and all occur at the same location further supporting this hypothesis (Beneloujaephajri et al., 2013, submitted). But the nature of the elicitors of these and corresponding receptors remain to be determined.
Sensing of stretch (or touch) by mechano-sensitive proteins, for example by stretch-activated channels in the membrane might be another way SMS is perceived and transduced. A possible model is that mechanical stimulus at the surface of the cell stretches such channels initiating a calcium flux [4, 16, 27, 28]. The SMS-induced transient burst of calcium (Figure 2A and B) and the induction of genes (Figure 2C) that were previously associated with the perception of mechanical stimuli  would argue in favor of this scenario. But SMS like cutinase- or wound-induced resistance to B. cinerea are independent of JA signaling [7, 20] (Figure 3), an observation that would differentiate SMS from a recent study on induced resistance to B. cinerea induced by leaf bending . Leaf bending (ten times) also referred to as gentle touch is only accompanied by a ca 30% reduction in lesion after inoculation with B. cinerea and is JA-sensitive . This contrasts with the present results where SMS was observed to lead to a full immunity to B. cinerea that is insensitive to JA. Experiments would now be needed with mutants blocked in mechano-sensitive touch receptors to differentiate between these pathways. It is most likely that SMS also leads to a major cellular reorganization such as that described by Hardham and colleagues (2008) ; the cellular details of the perception and attending mechanisms await now further studies.
Arabidopsis thaliana was grown on a pasteurized soil mix of humus and Perlite (3:1) in a growth chamber with a 12 h day/night photoperiod at 21°C/19°C, with a light intensity of 100 μE m−2 sec−1 and with a relative humidity of 60-70%. WT plants were the Arabidopsis accession Col0 obtained from the Arabidopsis Biological Research Center (Colombus, OH, USA). The Arabidopsis mutant referred to as gl1 was in the Col0 background . The Arabidopsis mutant myb96-1 was in the Col0 background and was previously described . The Arabidopsis mutants dde 2.2, opr3, coi 1.16 were in the Col0 background.
B. cinerea strain BMM, provided by Brigitte Mauch-Mani (University of Neuchâtel, Switzerland), were grown on Difco Potato Dextrose Agar (PDA) 39 g l−1 (Becton Dickinson, http://www.bd.com). Spores were harvested in water and filtered through glass wool to remove hyphae. Before inoculation, spores were diluted in ¼ strength Difco Potato Dextrose Broth (PDB) at 6 g l−1 (Becton Dickinson, http://www.bd.com) to the final concentration of 5 × 104 spores ml-1. Six μl of spore suspension were deposited on leaves of 4-week-old plants. Lesion diameter was measured 3 days after inoculation using the digital caliper series 500 (Mitutoyo, http://www.mitutoyo.com). Data were integrated via the software for metrology IBREXDLL (IBRit, http://www.ibr.com). The inoculated plants were kept 3 days under high humidity (covered trays) in the growth chamber. Fungal structures and dead plant cells were stained by boiling inoculated leaves for 5 min in a solution of alcoholic lactophenol trypan blue. Stained leaves were extensively cleared in chloral hydrate (2.5 g ml−1) at room temperature by gentle shaking, and then observed using a Leica DMR microscope with bright-field settings.
Plant leaves were gently rubbed between thumb and forefinger without pressing with the thumb. For the time course experiment the SMS treatment was repeated 1, 5, 7 and 10 successive times, for the others experiments the SMS treatment was repeated 10 successive times. For SMS treatment of entire leaves, the SMS treatment was carried out on both sides of the main vein. SMS treatment leaves were incubated in covered trays at high humidity (referred to as humid conditions); in some cases the trays were left uncovered after SMS treatment (referred to as dry conditions) at the same laboratory conditions. Inoculation with B. cinerea was performed within 10 min after SMS treatment, by placing a droplet of spores on the SMS-treated site.
For the collection of diffusates, 8 μl of ¼ PDB were incubated for 24 h on non-treated and SMS-treated WT and mutants leaves. Leaf diffusates were collected and mixed with Botrytis cinerea spores to the final concentration of 5 × 104 spores ml-1. The WT plants were inoculated in the same conditions as previously described.
ROS were detected using the fluorescent probe 5-(and-6)-carboxy-2’,7’-dichloro dihydrofluorescein diacetate (DCF-DA) (Sigma-Aldrich, http://www.sigmaaldrich.com) as previously described  . SMS treated- and non-treated leaves were vacuum-infiltrated (3 × 3 min) in 60 μM of DCF-DA in a standard buffer (1 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM 2-morpholinoethanesulfonic acid adjusted to pH6.1 with NaOH) . Leaves were then rapidly rinsed in DCF-DA buffer and observed using a Leica DMR epifluorescence microscope with a GFP filter set (excitation 480/40 nm, emission 527/30 nm) (Leica, http://www.leica.com). Microscope images were saved as TIFF files and processed for fluorescence quantification with Image J version 1.45 (NIH). Software settings were kept the same for every image analyzed and the area of green fluorescence corresponding to ROS production was expressed in pixels.
Chlorophyll extraction and quantification was performed according to a previously described protocol . Leaves were cut at the petiole, weighed and immersed in 30 ml of 80% ethanol. Chlorophyll was extracted in the dark at room temperature with gentle agitation. Aliquots were removed at 10, 20, 30, 40, 50 and 60 min after immersion. The chlorophyll content was determined by measuring absorbance at 647 and 664 nm and the micromolar concentration of total chlorophyll per gram of fresh weight of tissue was calculated from the following equation: (19.53 × (A647 nm) + 7.93 × (A664 nm))/g fresh weight. The toluidine blue test was carried out by placing 6 μl droplets of a 0.025% toluidine blue solution in ¼ PDB on the leaf surface. After incubation for 2 h, leaves were washed gently with distilled water to remove excess of the toluidine blue solution from leaves. For staining with Calcofluor white, leaves were bleached in absolute ethanol overnight, equilibrated in 0.2 M NaPO4 (pH 9) for 1 h, and incubated for 1 min in 0.5% Calcofluor white in 0.2 M NaPO4 (pH 9). Leaves were rinsed in NaPO4 buffer to remove excess of Calcofluor white and viewed under UV light on a GelDoc 2000 system (Biorad, http://www.biorad.com).
The non-treated and SMS-treated leaves (10X) were ground. RNA was prepared using the Trizol reagent containing 38% saturated phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate and 5% glycerol. RNA (1 μg) was then retrotranscribed into cDNA (Omniscript® RT kit, Qiagen, http://www.qiagen.com). RT-PCR was performed using Sensimix™ SYBR Green Kit (Bioline, http://www.bioline.com). Gene expression values were normalized to expression of the plant gene At4g26410, previously described as a stable reference gene . The primers used were TCH3fw 5′- TCAAGGTCAGGGTCAAGTGC; TCH3rev 5′- TTGGCGAAGCGATGATATTGC; TCH4fw 5′- GAAACTCCGCAGGAACAGTC; TCH4rev 5′- TGTCTCCTTTGCCTTGTGTG; CML24fw 5′- GAGTAATGGTGGTGGTGCTTGA; CML24rev 5′- ACGAATCATCACCGTCGACTAA; CML39fw 5′- GATTGCATTACTCCGGGGAG; CML39rev 5′- GAGGGCGAACTCATCAAAGC.
The monitoring of the cytoplasmic calcium concentrations change was performed using transgenic A. thaliana plants expressing aequorin under the control of the cauliflower mosaic virus promoter 35S (gift from Marc Knight, Durham University). Leaves from 4 weeks old aequorin expressing plants were incubated overnight in 10 μM of coelenterazine (CTZ) in the dark to allow the binding between CTZ and aequorin. Basal level and stability of the luminescence signal before SMS treatment were then assessed by introducing the leaves in the luminometer (Sirius single tube luminometer, Berthold detection system, http://www.berthold-ds.com) where luminescence values were immediately scored every 3 seconds for one minute. After this time, 5 events of SMS were applied to the leaves directly in the luminometer and reading was carried out for three minutes. The luminescence was detected by using FB12 Sirius PC software.
Whole leaves of 16- to 21-day-old plants expressing cytoplasmic localized cameleon YC3.6 were placed in an open top chamber . Leaves were imaged in vivo by an inverted fluorescence microscope Nikon Ti-E (Nikon, JP, http://www.nikon.com) with CFI planfluor 4× A.N.0,13 dry objective. Excitation light was produced by a fluorescent lamp Prior Lumen 200 PRO (Prior Scientific, UK) at 440 nm (436/20 nm) for Cameleon. Images were collected with a Hamamatsu Dual CCD Camera ORCA-D2 (Hamamatsu, Photonics, JP). The FRET CFP/YFP optical block A11400-03 (Emission 1 483/32 nm for CFP and Emission 2 542/27 nm for cpVenus with a dichroic mirror 510 nm) (Hamamatsu, Photonics, JP) was used for the simultaneous CFP and cpVenus acquisitions. Exposure time was 400 ms with a 2 × 2 CCD binning and images where acquired every 2 sec. Filters and dichroic mirror were purchased from Chroma (Chroma Technology Corporation, USA). The NIS-Element (Nikon, JP) was used as platform to control microscope, illuminator, camera and post-acquisition analyses. The fluorescence intensity was determined over regions of interest (ROIs) that correspond to the SMS-treated site. The SMS treatment was made 1 time. Due to the size of the imaged areas, the background was not subtracted. For cameleon analysis cpVenus and CFP emissions of the analyzed ROIs were used for the ratio (R) calculation (cpVenus/CFP) and normalized to the initial ratio (R0) and plotted versus time (ΔR/R0).
SMS-treated and non-treated surfaces leaves were viewed with an S-3500 N variable pressure scanning electron microscope from Hitachi (http://www.hitachi.com), equipped with a cold stage.
We thank Linda Grainger and Thérèse Mandel for their invaluable technical assistance. This work was made possible by funds to J.P.M. from the Swiss National Science Foundation (grant 125370) and from the Italian Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR) through the grant FIRB 2010 (RBFR10S1LJ_001) to A.C. We wish to thank Prof Chung-Mo Park (Seoul National University) for making the myb96-1 mutant available to us.
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