Production of reactive oxygen species (ROS) triggered by flg22 and Harpin.

<p>Time-course of ROS accumulation monitored by dihydrorhodamine 123 (DHR 123) in response to the water control (open circles), flg22 (1 µM, closed squares), or Harpin (9 µg^.ml⁻¹, closed triangles) in <i>V. rupestris</i> (<b>A</b>) versus <i>V. vinifera</i> cv. ‘Pinot Noir’ (<b>B</b>). Relative fluorescence recorded at constant exposure time (100 ms) was quantified relative to the respective base fluorescence by the Image J software as described in Material and Methods.</p

A simplified model for defence triggered by flg22 and Harpin in grapevine cells.

<p>Details are explained in the discussion. <b>Flg</b>, flg22; <b>Hrp</b>, bacterial protein Harpin; <b>flgr</b>, flg22 receptor (grapevine homologue of AtFLS2); <b>msc</b>, mechanosensitive ion channel; <b>MTs</b>, microtubules; <b>mAFs</b>, membrane-associated actin filaments; <b>Rboh</b>, grapevine homologue of NADPH dependent oxidase responsible for apoplastic oxidative burst (<b>ROS_ex</b>) that can permeate the plasma membrane (<b>ROS_int</b>); <b>MAPK</b>, MAPK-signalling pathway; <b>StSy</b>, stilbene synthase gene; <b>iAFs</b>, intracellular actin filaments; <b>Res</b>, <i>trans</i>-resveratrol; <b>δ-Vin</b>, δ-viniferin; <b>Pic</b>, <i>trans</i>-piceid.</p

Expression of defence-related genes induced by flg22.

<p><b>A, B</b> Representative gels showing transcript abundance followed by semi-quantitative RT-PCR after elicitation with 1 µM flg22 (<b>A</b>), and quantification relative to elongation factor 1α (<b>B</b>) as reference. The data represent mean values from three independent experimental series; error bars show standard errors. Genes of interest encode proteins including PAL, phenylalanine ammonium lyase; CHS, chalcone synthase; StSy, stilbene synthase; RS, resveratrol synthase; and CHI, chalcone isomerase; pathogenesis-related proteins: PR10 ad PR5, and PGIP: polygalacturonase-inhibiting protein. <b>C, D</b> Influence of MAPK signalling on the abundance of <i>StSy</i> transcripts. Cells were challenged by 1 µM flg22, by 9 µg^.ml⁻¹ Harpin (both in the solvent DMSO) alone or in combination with the MAPK cascades inhibitor PD98059 (PD). A representative agarose gel is shown in <b>C</b>, the quantification relative to elongation factor 1α from four independent experimental series in <b>D</b>, error bars represent standard errors.</p

Defence Signalling Triggered by Flg22 and Harpin Is Integrated into a Different Stilbene Output in <em>Vitis</em> Cells

<div><p>Plants can activate defence to pathogen attack by two layers of innate immunity: basal immunity triggered by pathogen-associated molecular pattern (PAMP) triggered immunity (PTI) and effector-triggered immunity (ETI) linked with programmed cell death. Flg22 and Harpin are evolutionary distinct bacterial PAMPs. We have previously shown that Harpin triggers hypersensitive cell death mimicking ETI in <em>Vitis rupestris</em>, but not in the <em>Vitis vinifera</em> cultivar ‘Pinot Noir’. In contrast, the bacterial PAMP flg22 activating PTI does not trigger cell death. To get insight into the defence signalling triggered by flg22 and Harpin, we compared cellular responses upon flg22 and Harpin treatment in the two <em>Vitis</em> cell lines. We found that extracellular alkalinisation was blocked by inhibition of calcium influx, and modulated by pharmacological manipulation of the cytoskeleton and mitogen-activated protein kinase activity with quantitative differences between cell lines and type of PAMPs. In addition, an oxidative burst was detected that was much stronger and faster in response to Harpin as compared to flg22. In <em>V. rupestris</em>, both flg22 and Harpin induced transcripts of defence-related genes including <em>stilbene synthase</em>, microtubule disintegration and actin bundling in a similar way, whereas they differed in <em>V. vinifera</em> cv. ‘Pinot Noir’. In contrast to Harpin, flg22 failed to trigger significant levels of the stilbene <em>trans</em>-resveratrol, and did not induce hypersensitive cell death even in the highly responsive <em>V. rupestris</em>. We discuss these data in a model, where flg22- and Harpin-triggered defence shares a part of early signal components, but differs in perception, oxidative burst, and integration into a qualitatively different stilbene output, such that for flg22 a basal PTI is elicited in both cell lines, while Harpin induces cell death mimicking an ETI-like pattern of defence.</p> </div

Response of the cytoskeleton to flg22.

<p><b>A</b> Disintegration of microtubules visualised by immunofluorescence1 h after addition of 1 µM flg22 or water as negative control. Size bar 20 µm. <b>B</b> Reorganisation of actin filaments visualised by FITC-phalloidin upon flg22 treatment as compared to the water control. Representative geometrical projections from Apotome Z-stacks collected from control (left) or after 3 h (flg22-induced, right) of treatment with 1 µM flg22 are shown. Size bar 20 µm. <b>C</b> Abundance of tyrosinylated α-tubulin in total extracts 24 h after additioin of 1 µM flg22 visualised by Western blotting probing with specific monoclonal antibodies. The same amount of total protein was loaded in each lane, verified by staining of a replicate by Coomassie Brilliant Blue. <b>D</b> Relative abundance of tyrosinylated α-tubulin quantified for the flg22 treatment (flg22, grey bars) as compared to control (con, white bars).</p

Stilbene accumulation in response to flg22 and Harpin.

<p>Cells of <i>V. rupestris</i> and <i>V. vinifera</i> cv. ‘Pinot Noir’ were exposed to either 1 µM flg22 or 9 µg^.ml⁻¹ Harpin for 0 (white bars) or 10 h (striped bars). Contents of <i>trans</i>-resveratrol, <i>trans</i>-piceid and δ-viniferin were determined by HPLC and quantified relative to their corresponding calibration curves based on reference standards, respectively. Mean values and standard errors from at least three independent experimental series are shown.</p

Modulation of harpin-triggered apoplastic alkalinisation by different auxins.

<p>Cells were treated with 9 μg ml^-1 harpin (hrp, closed circles) as a positive control, harpin combined with 10 μM (open triangles) or 50 μM auxins (IAA, NAA, or 2, 4-D, closed triangles), or ethanol used as solvent control (con, open circles) in <i>V</i>. <i>rupestris</i> (<b>A</b>, <b>C</b>, and <b>E</b>) and cv. ‘Pinot Noir’ (<b>B</b>, <b>D</b>, and <b>F</b>). Representative experiments from five biological replicas are depicted. Harpin and auxin were added at time 0, if measured isolated, for the combinations, auxins were added 1 h prior to harpin.</p

Working model on the antagonistic interaction of signalling triggered by harpin and auxin.

<p>To reduce complexity, only the earliest events are depicted, omitting ROS activation of calcium influx and effects of rac1-signalling on auxin transport. ① harpin activates the NADPH-dependent oxidase RboH leading to the production of superoxide that can spread in the apoplast. ② Superoxide can penetrate through the plasma membrane (probably by aquaporins) and glutathionylate actin in residue Cys374. This will sequester G-actin from being integrated into the growing end of actin filaments. ③ Alternatively, superoxide can be recruited to transduce the effect of auxin (perceived via the auxin-binding protein, ABP) upon the activation of phospholipase D (PLD) through the small G-protein Rac. ④ PLD will generate phosphatidic acids (PA) that can sequester actin capping proteins (cap) to the membrane, such that elongation of actin filaments is enabled. Alternatively, PA can be partitioned to recruit Rac for the activation of the RboH complex. In this case, the capping proteins will not be recruited to the membrane and constrain the elongation of actin filaments leading as secondary consequence to the formation of thick cables through the activity of severing proteins in combination with free G-actin the formation. ⑤ As third alternative, PA can be converted to PIP2, which will recruit actin-depolymerization factor (ADF) to the membrane. Since ADF is sustaining the monomer turnover at the minus end of actin filaments, this recruitment results in a higher stability of fine cortical actin filaments. The molecular targets for the inhibitors diphenyliodonium (DPI), and <i>n</i>-butanol are inserted in red. Hypothetical aspects of the model that have not been addressed experimentally in plant cells, are indicated by blue question marks: The interaction of Harpin with RboH (①) has not been addressed experimentally so far. Also the glutathionylation of actin in consequence of superoxide penetration (②), so far has been shown for animal systems, but not been addressed in plant cells.</p

Effect of Latrunculin and auxins on harpin-induced changes of cell viability.

<p>Cells were treated with Latrunculin B (Lat B, 2 μM), with harpin (hrp, 9 μg ml^-1), or with LatB in combination with harpin and auxin (IAA, NAA or 2, 4-D, 50 μM) following subculture weekly, versus ethanol as solvent control (con) in <i>V. rupestris</i> (A) and cv. ‘Pinot Noir’ (B). Data show mean and standard errors from three independent experiments with 500 cells. Significance levels of differences was analyzed using ANOVA with * significant at P = 5%, and ** significant at P = 1%.</p

Quantification of actin responses by measuring apparent width (w) of actin bundles.

<p><b>A</b> Time course of actin bundling in response to 9 μg^.ml^-1 harpin. <b>B</b> Dose-dependency of harpin-induced actin bundling over different concentrations of either the natural auxin indole-3-acetic acid (IAA), or the artificial auxins 1-napthyl acetic acid (NAA), or 2,4-dichlorophenoxy acid (2,4-D) as compared to the control (con) or harpin (hrp) in the absence of supplemental auxins measured after 60 min of incubation in <i>V</i>. <i>rupestris</i> and cv. ‘Pinot Noir’. Concentrations of auxins indicated in μM. "n.d." means "not determined" to indicate that this value was not measured in cv. ‘Pinot Noir’ (to distinguish it from a zero value). <b>C</b> Effect of pharmacological inhibition of phospholipase D by preincubation with 0.5% (v/v) of <i>n</i>-butanol or the inactive analogue <i>sec</i>-butanol for 30 min prior to the indicated treatment for 30 min, and effect of pharmacological inhibition of RboH on harpin-induced bundling by preincubation with 20 μM dephenylene iodonium (DPI) for 30 min prior to the indicated treatment for 30 min. <b>IAA</b> 10 μM indole-3-acetic acid, <b>hrp</b> 9 μg^.ml^-1 harpin. Arrows represent complete elimination of actin, such that no value for bundling could be measured. <b>n</b> represents the number of individual cells per sample, error bars standard errors for a population randomly collected from three biological replicates and 200 cells were used for each experiment. Brackets indicate significance levels of differences with * significant at <i>P</i> = 5%, and ** significant at <i>P</i> = 1%. "n.d." means "not determined" to indicate that this value was not measured (to distinguish it from a zero value).</p