Fig. 4: Stochastic and spatially structured time-course distribution of flagellar and T3SS expression within apoplastic microcolonies.
a, CLSM Z-stack compilation at different dpi showing apoplastic microcolonies of hopAB1::GFP3 fliC::tdT bacteria. All images examined (n = 5 or more) per biological replicate (n = 2) and independent experiment (n = 2 for 1 and 2 dpi; n = 4 for 3 and 4 dpi). b, Images from a including orthogonal projections. The colored lines within the central images indicate the planes that generate each orthogonal section: green lines indicate the section that is displayed in the green-boxed images (images above per time point), red lines indicate the section that is displayed in the red-boxed images (images on the right per time point). In green- or red-boxed images, the blue line correspondes to the section displayed in the central image. Scale bars, 5 µm. c, Schematic leaf 3D structure with close-up showing location of apoplastic microcolonies. Position and orientation of Z-stack acquisition are indicated; 1–3 represent three planes of a bacterial microcolony growing from the lower epidermis inner surface inwards (1 closest to cell surface and 3 furthest) as shown in d and Supplementary Video 8. Plane 4 cuts through a bacterial microcolony growing wrapped around a spongy mesophyll cell (SMC) (as microcolony and frame shown in f and Z-stack shown in Supplementary Video 9). d, Frames of a Z-stack acquisition taken from an apoplastic microcolony of hrpL::GFP3 fliC::tdT bacteria at 4 dpi. Contrast and brightness were adjusted to improve visualization and kept constant throughout. Scale bars, 10 µm. Lower epidermis and mesophyll indicate relative position of frames within the leaf. e, Schematic distribution of fluorescence for fliC::tdT and T3SS::GFP3 within the type of apoplastic microcolonies indicated in c (1–3) and shown in d: planes closest to lower epidermis (abaxial side) show predominantly green bacteria and those furthest predominantly red, with intermediate plane displaying yellow. Heterogeneity is observed within each plane. f, Z-stack frame taken from an apoplastic microcolony of hopAB1::GFP3 fliC::tdT bacteria (Z-stack in Supplementary Video 9) from experiments described in a. Scale bars, 20 µm. g, Schematic fluorescence distribution for fliC::tdT T3SS::GFP3 bacteria within apoplastic microcolonies of the type indicated in c (4) and shown in f: bacteria closest to the cell are predominantly green, turning to yellow and red as they get further away. Heterogeneity is displayed throughout. In e and g, legend indicates green as T3SSON/ FlagellaOFF bacteria, red as T3SSOFF/ FlagellaON and yellow/orange as bacteria expressing both to different levels.
