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

Figure EV2. Fo5176‐induced depletion of the cellulose synthase machinery and acidification of the plasma membrane interface, measured with newly designed sensors, can be buffered.

Figure EV2

  1. Representative images of 6‐day‐old WT (Col‐0) root epidermal and cortex cells expressing apo‐pHusion or SYP122‐phusion. Much less intracellular signal of pHusion is observed for SYP122‐pHusion than for apo‐pHusion. Scale bar = 20 μm.
  2. In vivo calibration of SYP122‐pHusion in 6‐day‐old roots. Dots represent individual samples with N ≥ 5 seedlings per standard buffer. Data points were fitted using sigmoidal regression.
  3. Representative images of 6‐day‐old WT (Col‐0) root epidermal and cortex cells expressing pHGFP‐Lti6b. Cell walls of seedlings were counterstained with 3 μg/ml propidium iodide to illustrate plasma membrane localization of pHGFP‐Lti6b of two adjacent cells (white arrow). Scale bars = 10 μm.
  4. In vivo calibration of pHGFP‐Lti6b in 6‐day‐old roots. Dots represent individual samples with N ≥ 5 seedlings per standard buffer. Data points were fitted using sigmoidal regression.
  5. Cytoplasmic pH variation of WT roots expressing the pHcyto free pHGFP sensor 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 ≥ 12 seedlings from three independent experiments. RM two‐way ANOVA on half MS + H2O versus half MS + elicitors: P = 0.42 (treatment), P = 0.06 (time), P = 0.08 (treatment × time).
  6. Apoplastic ∆pH of WT roots expressing the pHapo SYP122‐pHusion sensor over time, either in half MS or in half MS + 5 mM MES. Imaging started 5 min before either H2O or a fungal elicitors were added (0 min). Values are mean ± SEM, N ≥ 16 seedlings from three independent experiments. RM two‐way ANOVA on half MS + H2O versus half MS + elicitors: P ≤ 0.001 (treatment), P ≤ 0.05 (time), P ≤ 0.001 (treatment × time).
  7. Cortical ∆pH of WT roots expressing the pHcortical pHGFP‐Lti6b sensor 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). Mixed‐effects model on half MS + H2O versus half MS + elicitors: P ≤ 0.01 (treatment), P ≤ 0.001 (time), P ≤ 0.001 (treatment × time).
  8. Apoplastic pH of WT roots expressing the pHapo SYP122‐pHusion sensor over time in half MS. Imaging started 5 min before either H2O (mock) or 1 mM chitin were 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 + chitin: P ≤ 0.05 (treatment), P ≤ 0.001 (time), P ≤ 0.001 (treatment × time). This chitin assay was done simultaneously to the elicitor treatments (Fig 2E); therefore, they share the same mock (H2O) curve.

Source data are available online for this figure.