Defence signalling triggered by Flg22 and Harpin is integrated into a different stilbene output in Vitis cells

Abstract

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 Vitis rupestris, but not in the Vitis vinifera 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 Vitis 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 V. rupestris, both flg22 and Harpin induced transcripts of defence-related genes including stilbene synthase, microtubule disintegration and actin bundling in a similar way, whereas they differed in V. vinifera cv. 'Pinot Noir'. In contrast to Harpin, flg22 failed to trigger significant levels of the stilbene trans-resveratrol, and did not induce hypersensitive cell death even in the highly responsive V. rupestris. 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.

Figure 1. Apoplastic alkalinisation evoked by flg22 and Harpin in the two grapevine cell lines.

A, B Dose response of extracellular alkalinisation to flg22 over time in Vitis rupestris (A) and Vitis vinifera cv. ‘Pinot Noir’ (B). C, D Analysis of the maximum change of extracellular pH in response to the increasing concentration of flg22. Data were fitted using a Michaelis-Menten equation [f (x) = ΔpH_max×x/(EC₅₀+x)], where ΔpH_max = 1.251 (V. rupestris) or 0.497 (V. vinifera cv. ‘Pinot Noir’), and EC₅₀ = approximately 4.825 nM (V. rupestris) or 876.86 nM (V. vinifera cv. ‘Pinot Noir’), respectively. E–H Role of Gd-sensitive calcium channels for apoplastic alkalinisation induced by 1 µM flg22 (E, G) or 9 µg^.ml⁻¹ Harpin (F, H) in combination with the solvent DMSO (open circles) or with 20 µM of GdCl₃ (closed circles) either in V. rupestris (E, F) or V. vinifera cv. ‘Pinot Noir’ (G, H), respectively. Representative timelines from five independent series are shown.

Figure 2. Feedback of downstream factors on flg22- and Harpin-induced apoplastic alkalinisation.

A–D Effect of the MAPK cascades inhibitor PD98059 (PD) on flg22- and Harpin-dependent alkalinisation in V. rupestris (A, B) versus V. vinifera cv. ‘Pinot Noir’ (C, D). Cells were elicited by either 1 µM flg22 (A, C) or 9 µg^.ml⁻¹ Harpin (B, D) in combination with 0 µM (open circles), 10 µM (closed triangles), or 100 µM (closed squares) PD98059 (PD). E–H Effects of cytoskeletal drugs on flg22 and Harpin-dependent alkalinisation, respectively. Effects of the microtubule inhibitor Oryzalin (+Ory, 20 µM, closed squares), or the actin inhibitor Latrunculin B (+LatB, 2 µM, closed triangles) in V. rupestris (E, F) versus V. vinifera cv. ‘Pinot Noir’ (G, H) were compared to the solvent control (DMSO, open circles). Representative timelines from five independent series are shown.

Figure 3. Production of reactive oxygen species (ROS) triggered by flg22 and Harpin.

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 V. rupestris (A) versus V. vinifera cv. ‘Pinot Noir’ (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.

Figure 4. Expression of defence-related genes induced by flg22.

A, B Representative gels showing transcript abundance followed by semi-quantitative RT-PCR after elicitation with 1 µM flg22 (A), and quantification relative to elongation factor 1α (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. C, D Influence of MAPK signalling on the abundance of StSy 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 C, the quantification relative to elongation factor 1α from four independent experimental series in D, error bars represent standard errors.

Figure 5. Stilbene accumulation in response to flg22 and Harpin.

Cells of V. rupestris and V. vinifera 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 trans-resveratrol, trans-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.

Figure 6. Response of the cytoskeleton to flg22.

A Disintegration of microtubules visualised by immunofluorescence1 h after addition of 1 µM flg22 or water as negative control. Size bar 20 µm. 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. C 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. D Relative abundance of tyrosinylated α-tubulin quantified for the flg22 treatment (flg22, grey bars) as compared to control (con, white bars).

Figure 7. Time course of cell mortality in response to flg22 and Harpin.

The relative frequency of dead cells after treatment with flg22 (1 µM, dotted bars) or Harpin (9 µg^.ml⁻¹, shaded bars) as compared to the water control (white bars) in V. rupestris (A) and V. vinifera cv. ‘Pinot Noir’ (B) was followed over time scoring samples of 1500 cells for each data point. Mean values and standard errors from four independent experimental series are shown.

Figure 8. A simplified model for defence triggered by flg22 and Harpin in grapevine cells.

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