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. 2019 Nov 18;38(24):e101822. doi: 10.15252/embj.2019101822

Figure 2. Fo5176‐induced depletion of the cellulose synthase machinery and acidification of the plasma membrane interface can be buffered and are dependent on upregulation of AHA activity.

Figure 2

  1. Representative image of a 5‐day‐old WT (Col‐0) GFP‐CesA3 and mCh‐TUA5 dual‐labeled root epidermal cell in half MS + 5 mM MES (left panel) or upon 5‐min elicitor treatment in half MS + 5 mM MES (right panel). Scale bar = 5 μm.
  2. GFP‐CesA3 density at the plasma membrane after elicitor treatment in half MS + 5 mM MES as depicted in (A). Box plots: centerlines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend to the minimum and maximum. N ≥ 35 cells from 10 roots and three independent experiments.
  3. Microtubule density at the cell cortex after elicitor treatment in half MS + 5 mM MES as depicted in (A). Box plots as described in (B). N ≥ 29 cells from 10 roots and three independent experiments.
  4. Representative surface plot of a WT root expressing the pHapo sensor SYP122‐pHusion grown in half MS (−5 to 0 min). At 0 min, either H2O (upper panel) or an elicitor mix (lower panel) was added. Upon elicitor treatment, the signal intensity in the depicted 488 nm channel drastically decreases (highlighted with a blue square).
  5. Apoplastic pH in WT roots expressing the pHapo sensor SYP122‐pHusion over time, either in half MS or in half MS + 5 mM MES. Imaging started 5 min before either H2O or a fungal elicitor mix was added (0 min). Values are mean ± SEM, N ≥ 15 seedlings from three independent experiments. RM two‐way ANOVA on half MS + H2O versus half MS + elicitors: P ≤ 0.05 (treatment), P ≤ 0.001 (time), P ≤ 0.001 (treatment × time).
  6. Cortical pH of WT roots expressing the pHcortical sensor pHGFP‐Lti6b over time, either in half MS or in half MS + 5 mM MES. Imaging started 5 min before either H2O or a fungal elicitor mix was added (0 min). Values are mean ± SEM, N = 16 seedlings from three independent experiments. Mixed‐effects model on half MS + H2O versus half MS + elicitors: P = 0.80 (treatment), P ≤ 0.001 (time), P ≤ 0.001 (treatment × time).
  7. Representative surface plot of WT root expressing the pHapo sensor SYP122‐pHusion grown 30 min in half MS (left panel) or half MS + Fo5176 hyphae (right panel). The hyphae treated root shows drastically reduced signal intensity in the depicted 488 nm channel (highlighted with a green square).
  8. Apoplastic pH of WT roots expressing the pHapo sensor SYP122‐pHusion over time, either in half MS or half MS + Fo5176 hyphae. Roots were exposed to hyphae for 5 min before imaging started. Values are mean ± SEM, N ≥ 12 seedlings from three independent experiments. RM two‐way ANOVA on half MS versus half MS + elicitors: P ≤ 0.01 (treatment), P ≤ 0.001 (time), P ≤ 0.001 (treatment × time).
  9. Cortical pH variation of WT roots expressing the pHcortical sensor pHGFP‐Lti6b over time, either in half MS or in half MS + Fo5176 hyphae. Roots were exposed to hyphae for 5 min before imaging started. Values are mean ± SEM, N ≥ 13 seedlings from three independent experiments. RM two‐way ANOVA on half MS versus half MS + elicitors: P ≤ 0.01 (treatment), P = 0.23 (time), P ≤ 0.001 (treatment × time).
  10. Western blots showing chemiluminescent signals of an anti‐pThr or anti‐AHA incubated membrane loaded with Arabidopsis root samples treated for 8 min with either half MS or half MS + Fo5176 hyphae. The Ponceau S panel shows total protein content. The AHA band used for quantification is indicated with an arrowhead. Dashed line separates different treatments of the same membrane.
  11. Relative AHA phosphorylation status from Western blots as shown in (J). Normalized signal intensity ratio of anti‐pThr in respect to anti‐AHA is shown. Box plots as described in (B). N = 5 independent experiments. Welch's unpaired t‐test; *P‐value ≤ 0.05.

Source data are available online for this figure.