Fig. 3: Flagellar and T3SS expression impact on bacterial growth.
a, Left: competitive index assay overview: inocula containing 1:1 mixes of wild-type and mutant (or plasmid-carrying) LB-grown cultures are used to inoculate leaves (in planta CI) or to start fresh cultures (in vitro CI). Resulting mixed cultures and apoplast-extracted bacteria (4 dpi) are serially diluted and plated with or without antibiotics for c.f.u. determination (output). Right: CI is calculated as mutant-to-wild-type c.f.u. ratio in output divided by ratio in input. CIs different from 1.0 indicate strains growing significantly differently: mutant outgrowing wild type (CI > 1.0) or vice versa (<1). b, CIs for mutants vs wild-type strain (WT) in HIM or LB (n = 3–9 as shown). c, HIM growth rates for ΔhrpL mutant and wild type. d, In planta CIs for wild type constitutively expressing hrpL (pHrpL) or ΔfliC, vs WT at 4 dpi (n = 10 as shown). Plants for ΔfliC CI pretreated with flg22. In b–d, data show mean ± s.e.m. Individual data for each biological replicate shown; different shades identify independent experiments, and results with asterisks are significantly different from 1.0 (non-parametric two-sided Student’s t-test). Significant P values indicated. e, Time-lapse images of fliC::GFP3 bacteria growing on agar pads: phase-contrast channel (top), GFP fluorescence (bottom). Contrast and brightness adjusted to improve visualization and kept constant across frames. f, Correlation between growth rates and fluorescence intensity of individual fliC::GFP3 cells. Each point represents a single cell, with fliC expression and growth rate averaged over cell lifetime. Grey-shaded area shows 95% confidence interval around linear regression model fit. Correlation tested with Pearson’s correlation coefficient using two-sided test (R = −0.17; P = 1.03 × 10−9). g, Growth rates for high-GFP- and low-GFP-signal cells (determined by splitting the population using median fluorescence intensity) show significantly slower growth in high-GFP cells (two-sided Mann–Whitney U-test, P = 3.6 × 10−8). Data presented as boxplots with first and third quartiles as bottom and top bounds, median shown as horizontal line in the box, and 1.5 interquartile range shown as whiskers. Outliers not shown. Grey violin plot shows distribution of all data points summarized in boxplot. White diamonds show mean growth rate for all cells considered.
