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. 2020 Nov 6;11:603693. doi: 10.3389/fpls.2020.603693

FIGURE 1.

FIGURE 1

A. thaliana COLORFUL-PR1pro reporter lines enable qualitative and quantitative monitoring of SA signaling responses at single-cell resolution. (A) Schematic representation of the COLORFUL-PR1pro T-DNA construct harboring the fluorescent modules PR1-VENUS-N7 (nuclear localized reporter), UBQ10-mKATE2-N7 (nuclear-localized reference) and CaMV35S-EGFP-LTI6b (plasma membrane marker). BastaR, selectable marker; LB/RB, left/right borders of T-DNA. (B) Maximum projections of CLSM z-stack images showing expression of reporter modules in leaves of 12-day-old transgenic A. thaliana line COLORFUL-PR1pro#1 at 24 h post incubation with mock solution (top) or 0.5 mM sodium salicylate (bottom); epidermal cells (cyan arrowhead/line), mesophyll cells (yellow arrowhead/line) and guard cells (orange arrowhead/line). (C) Side-view 3D projections of the CLSM z-stack images presented in b show epidermis and mesophyll cell-specific distribution and intensities of reporter- and reference-associated fluorescence signals (left and middle) and their overlays (right). XZ Scale bar = 25 μm. (D,E) z-depth of reporter (left) and reference (right) fluorescence signals from mock (D) and SA (E) treatments shown in b. Scale bar, 25 μm. (F,G) Automated nuclear fluorescence detection and quantification using the COLORFUL SPOTTER plugin. CLSM z-stack images corresponding to epidermal (F) and mesophyll (G) cell layers from the SA treatment experiment presented in B (bottom) were used separately for generation of tissue-specific maximum projections of reporter (left) or reference (right) fluorescence. Subsequent COLORFUL SPOTTER-mediated processing identifies nuclear fluorescence signals, generates a corresponding fluorescence detection mask (yellow overlay), assigns cell-specific index numbers, and quantifies individual fluorescence intensities (H,I). White X indicates manually excluded spots resulting from erroneous merging of close nuclear signals. Scale bar, 25 μm. (H,I), COLORFUL SPOTTER-mediated quantification of single-cell reporter and reference fluorescence signals from f and g, respectively. (J) Reporter activities in leaf guard, pavement and palisade mesophyll cells of wild-type (WT) and npr1-1 mutant plants after treatment of seedlings with 0.5 mM sodium salicylate for 24 h. Box plots show first quartile (lower line); median (center line); mean (+); third quartile (upper line); whiskers extend 1.5 times the interquartile range, and outliers (dots), n = 9–10. Data are relative to mock of WT. Different letters indicate significant differences (Two-way ANOVA followed by Tukey’s multiple comparison test, p < 0.05; see also Supplementary Figures S1, S2A, S4A).