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

Subcellular compartmentalisation of nicotine / nornicotine biosynthetic enzymes—modified from [13].

<p>A622: isoflavone reductase-like protein; ADC: arginine decarboxylase; BBL: berberine bridge enzyme-like; MPO: N-methylputrescine oxidase; NND: nicotine <i>N</i>-demethylase; ODC: ornithine decarboxylase; PMT: putrescine methyltransferase; QPT: quinolinate phosphoribosyltransferase. Genes that have been overexpressed in the present study are shown in green.</p

Nicotine and nornicotine contents in senescent leaves of different <i>Nicotiana</i> species and metabolic impact of overexpressed <i>Nom</i>CYP82E4-GFP.

<p><b>(a)</b> The low ratio of nornicotine to nicotine in <i>N</i>. <i>tabacum</i> and the high level of nornicotine compared to nicotine in <i>N</i>. <i>tomentosiformis</i> are evident. Error bars represent SE from three independent experiments. <b>(b, c)</b> Alkaloid profiles measured after 3 days of culture in presence of 100 μM jasmonic acid either intracellularly <b>(b)</b> or secreted to the medium <b>(c)</b>. The levels of nornicotine below detection limit is indicated by non-detectable (N.D.). Note the difference in scales between <b>(b)</b> and <b>(c)</b> (the level of secreted alkaloids is in some case around tenfold lower). For the alkaloid measurement, mean and SE are shown from six independent experimental series. Significant differences to the non-transformed WT cells assessed by a Student’s t-test are indicated by * (<i>P</i> < 0.05) or ** (<i>P</i> < 0.01), respectively.</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