ILEE library enables the discovery of actin dynamic features of bacteria infected leaf tissue. Leaves of the Arabidopsis actin marker line Col-0/GFP-fABD2 were inoculated with mock or a virulent bacterial pathogen, Pseudomonas syringae pv. tomato DC3000 with empty vector (EV, identical to wild type); images (n = 28 for mock, n = 31 for EV) of epidermal cells were captured by laser scanning confocal microscopy at 24 h post-inoculation (hpi). MGT and ILEE were applied to generate binary images and all indices were computed using ILEE_CSK. Double-blinded samples were provided to three operators (experienced researchers) OA, OB, and OC for comparison. OA additionally provides data using a universal threshold (OA-fix). (a) Experimental schematic diagram. (b) Corelative comparison of MGTs of individual samples determined by different operators. A very low correlation between each pair of operators indicates MGT has an increased risk of bias and is potentially inaccurate. (c–h, l, m, and n) Output indices occupancy, linear density, skewness, CV, diameter_TDT, diameter_SDT, segment density, static branching activity, and local anisotropy, respectively. Error bar = standard deviation. Multiple comparisons are conducted using a two-sided t test without family-wise error correction (because image samples themselves are ground truths, and data are exact measurements of methods, rather than approximations). Groups without overlapping letters have a P-value lower than 0.05. Data distribution was assumed to be normal but not formally tested. For n, a visual illustration of the concept of local anisotropy is attached, where each red line segment points to the “averaged” direction of actin growth in the local area and the length shows the intensity of consistency of the direction. (i, j, and k) Comparisons of different indices in bundling class by individual image samples. The linear correlations between bundling indices are plotted and measured by Pearson coefficient. Skewness and CV have medium-weak correlation; diameter_TDT and diameter_SDT have strong correlation; diameter_SDT, as a representative of direct indicator, and CV, as a representative of indirect indicator, have literally no correlation. “r” represents Pearson correlation coefficient; “p” represents the possibility to reject the null-hypothesis of no existent correlation by two-sided t test. Data distribution was assumed to be normal but not formally tested.
Figure 6.

ILEE library enables the discovery of actin dynamic features of bacteria infected leaf tissue. Leaves of the Arabidopsis actin marker line Col-0/GFP-fABD2 were inoculated with mock or a virulent bacterial pathogen, Pseudomonas syringae pv. tomato DC3000 with empty vector (EV, identical to wild type); images (n = 28 for mock, n = 31 for EV) of epidermal cells were captured by laser scanning confocal microscopy at 24 h post-inoculation (hpi). MGT and ILEE were applied to generate binary images and all indices were computed using ILEE_CSK. Double-blinded samples were provided to three operators (experienced researchers) OA, OB, and OC for comparison. OA additionally provides data using a universal threshold (OA-fix). (a) Experimental schematic diagram. (b) Corelative comparison of MGTs of individual samples determined by different operators. A very low correlation between each pair of operators indicates MGT has an increased risk of bias and is potentially inaccurate. (c–h, l, m, and n) Output indices occupancy, linear density, skewness, CV, diameter_TDT, diameter_SDT, segment density, static branching activity, and local anisotropy, respectively. Error bar = standard deviation. Multiple comparisons are conducted using a two-sided t test without family-wise error correction (because image samples themselves are ground truths, and data are exact measurements of methods, rather than approximations). Groups without overlapping letters have a P-value lower than 0.05. Data distribution was assumed to be normal but not formally tested. For n, a visual illustration of the concept of local anisotropy is attached, where each red line segment points to the “averaged” direction of actin growth in the local area and the length shows the intensity of consistency of the direction. (i, j, and k) Comparisons of different indices in bundling class by individual image samples. The linear correlations between bundling indices are plotted and measured by Pearson coefficient. Skewness and CV have medium-weak correlation; diameter_TDT and diameter_SDT have strong correlation; diameter_SDT, as a representative of direct indicator, and CV, as a representative of indirect indicator, have literally no correlation. “r” represents Pearson correlation coefficient; “p” represents the possibility to reject the null-hypothesis of no existent correlation by two-sided t test. Data distribution was assumed to be normal but not formally tested.

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