The cytoskeleton enhances gene expression in the response to the Harpin elicitor in grapevine

Abstract

The cytoskeleton undergoes dramatic reorganization during plant defence. This response is generally interpreted as part of the cellular repolarization establishing physical barriers against the invading pathogen. To gain insight into the functional significance of cytoskeletal responses for defence, two Vitis cell cultures that differ in their microtubular dynamics were used, and the cytoskeletal response to the elicitor Harpin in parallel to alkalinization of the medium as a fast response, and the activation of defence-related genes were followed. In one cell line derived from the grapevine cultivar 'Pinot Noir', microtubules contained mostly tyrosinylated alpha-tubulin, indicating high microtubular turnover, whereas in another cell line derived from the wild grapevine V. rupestris, the alpha-tubulin was strongly detyrosinated, indicating low microtubular turnover. The cortical microtubules were disrupted and actin filaments were bundled in both cell lines, but the responses were elevated in V. rupestris as compared with V. vinifera cv. 'Pinot Noir'. The cytoskeletal responsiveness correlated with elicitor-induced alkalinization and the expression of defence genes. Using resveratrol synthase and stilbene synthase as examples, it could be shown that pharmacological manipulation of microtubules could induce gene expression in the absence of elicitor. These findings are discussed with respect to a role for microtubules as positive regulators of defence-induced gene expression.

Fig. 1.

Cell morphology and response of pH to Harpin in cv. ‘Pinot Noir’ (A, C, E) and V. rupestris (B, D, F). (A, B) Cell morphology in differential interference contrast. Size bar 50 μm. (C, D) Representative time courses of the pH response to 0, 9, and 90 μg ml⁻¹ Harpin. (E, F) Cumulative analysis of time courses in responses to increasing concentrations of Harpin. The data were fitted using a Michaelis–Menten function. TpH₅₀ represents the time to reach 50% of the maximal response. The curves represent the average from n ≥15 individual time courses. (This figure is available in colour at JXB online.)

Fig. 2.

Response of cortical microtubules to Harpin in cv. ‘Pinot Noir’ (A, B) and V. rupestris (C, D). Representative geometrical projections from z-stacks collected prior to (A, C) or after 3 h (B, D) of treatment with 9 μg ml⁻¹ Harpin. Microtubules were visualized by immunofluorescence. Size bar=50 μm. (E) Method to quantify microtubule density (as a measure of microtubule integrity) as integrated fluorescence along a probing line. (F) Microtubule density in relative units prior to (open bars) or after 3 h of treatment with 9 μg ml⁻¹ Harpin (striped bars). Error bars represent standard errors. The values summarize the data of 18–39 individual cells collected from at least three independent experiments. (G) Relative abundance of tyrosinylated α-tubulin (recorded by the ATT antibody) versus detyrosinated α-tubulin (recorded by the DM1A antibody) in total extracts from cv. ‘Pinot Noir’ or V. rupestris that had been either raised under control conditions or challenged for 16 h with 9 μg ml⁻¹ Harpin. The same amount of total protein was loaded in each lane. (This figure is available in colour at JXB online.)

Fig. 3.

Response of actin filaments to Harpin in cv. ‘Pinot Noir’ (A, B) and V. rupestris (C, D). Representative geometrical projections from z-stacks collected prior to (A, C) or after 3 h (B, D) of treatment with 9 μg ml⁻¹ Harpin. Actin filaments were visualized by fluorescence-labelled phalloidin. Size bar=20 μm. (This figure is available in colour at JXB online.)

Fig. 4.

Response of defence-related genes to Harpin in cv. ‘Pinot Noir’ and V. rupestris. (A) Position of the analysed enzymes in flavonoid and stilbene metabolism. (B) Representative time courses of transcript abundance followed by RT-PCR in response to 9 μg ml⁻¹ Harpin. The upper group represents genes of the flavonoid and stilbene pathway: PAL, phenylalanine ammonium lyase; CHS, chalcone synthase; StSy, stilbene synthase; RS, resveratrol synthase; and CHI, chalcone isomerase. The middle group represents pathogenesis-related genes: PR10, pathogenesis-related protein 10; and PGIP, polygalacturonase-inhibiting protein. Elongation factor 1α (EF) was used as internal standard for quantification. (C) Time course of transcript abundance for RS and StSy relative to elongation factor factor 1α. The data represent averages from three independent experimental series; error bars represent standard errors. (D) Expression of RS and StSy relative to untreated controls after pre-treatment with anticytoskeletal compounds for 30 min and allowing the response to be expressed for an additional 2 h. Concentrations were 1 μM for phalloidin (Pha), 1 μM for latrunculin B (LatB), 10 μM for cytochalasin D (CytD), 10 μM for oryzalin (Ory), 10 μM for taxol (Tax), and 1% for DMSO as solvent control for the oryzalin experiment. The data represent averages from three independent experimental series; error bars represent standard errors. * different from the untreated control significant at the 95% confidence level, ** different from the untreated control significant at the 99% confidence level.