Live imaging of root-bacteria interactions in a microfluidics setup

Abstract

Plant roots play a dominant role in shaping the rhizosphere, the environment in which interaction with diverse microorganisms occurs. Tracking the dynamics of root-microbe interactions at high spatial resolution is currently limited because of methodological intricacy. Here, we describe a microfluidics-based approach enabling direct imaging of root-bacteria interactions in real time. The microfluidic device, which we termed tracking root interactions system (TRIS), consists of nine independent chambers that can be monitored in parallel. The principal assay reported here monitors behavior of fluorescently labeled Bacillus subtilis as it colonizes the root of Arabidopsis thaliana within the TRIS device. Our results show a distinct chemotactic behavior of B. subtilis toward a particular root segment, which we identify as the root elongation zone, followed by rapid colonization of that same segment over the first 6 h of root-bacteria interaction. Using dual inoculation experiments, we further show active exclusion of Escherichia coli cells from the root surface after B. subtilis colonization, suggesting a possible protection mechanism against root pathogens. Furthermore, we assembled a double-channel TRIS device that allows simultaneous tracking of two root systems in one chamber and performed real-time monitoring of bacterial preference between WT and mutant root genotypes. Thus, the TRIS microfluidics device provides unique insights into the microscale microbial ecology of the complex root microenvironment and is, therefore, likely to enhance the current rate of discoveries in this momentous field of research.

Keywords: Bacillus subtilis; TRIS; live-imaging microscopy; microbial community dynamics; root–bacteria interaction.

Fig. 1.

TRIS: a microfluidic device for tracking root–bacteria interactions. (A) Illustration of the TRIS device mounted on the microscope stage (dark rim). (Inset) Schematic longitudinal section of a microfluidic channel containing root and bacterial cells (red; not drawn to scale). (B) Top view of Arabidopsis seedlings growing in plastic pipette tips attached to the TRIS device. Roots are visible as thin white lines extending from tip ends. (Scale bar: 1 cm.) (C) Microscopic view of nine Arabidopsis roots growing inside the TRIS microfluidic device captured using bright-field illumination at 10× magnification. Arrows: a, inlet port.; b, tip in a root-dedicated port with c, root extending toward the outlet port; d, outlet port. (Scale bar: 5 mm.)

Fig. S1.

Mask design for generating the TRIS microfluidic device. Microfluidic device mask design used for the photolithography process. (A) Array of nine single microfluidic channels. *Magnification of one single-channel diminutions. (B) Array of six double microfluidic channels. **Magnification of one double-channel diminutions. Permeable divider indicated by the black arrow.

Fig. S2.

The TRIS microfluidic device placed on a microscope stage. View of the TRIS device mounted onto a microscope stage. The device contains microfluidics channels with Arabidopsis seedlings, and the entire setup includes a tubing system, 1-mL syringes, a medium reservoir, and a humidity chamber made of acrylic plastic. (Scale bar: 5 cm.)

Fig. 2.

Live imaging of B. subtilis cells interaction with Arabidopsis root. (A) Accumulation of B. subtilis cells near the root elongation zone of Arabidopsis seedlings (white arrows) during the first 30 min of coincubation. Each image is an MIP from a short video (11 s; ∼100 frames) obtained using dark-field microscopy. The rapid accumulation suggests a chemotactic response of bacterial cells toward high local concentrations of root exudates. (B) Selected images from time-lapse confocal microscopy of a WT Arabidopsis root (bright field) incubated with red fluorescent-labeled B. subtilis (mKate) for 12 h. Images are representative of nine independent inoculation experiments. (C) A B. subtilis biofilm defective mutant (eps) shows similar attraction as the WT strain but with little or no subsequent biofilm formation. Images are representative of four independent inoculation experiments. The white area plots (on the right side of the mKate fluorescent images) show normalized bacterial quantification. These area plots could be compared between the experiments in B and C; x axes in all area plots have the same length and range between zero and one normalized intensity. (Insets in B and C) Higher magnification view of the area marked by a dashed box in the mKate view. The same area is marked by a dashed box in the bright-field view. Magnification in B shows biofilm formation at a mature part of the root compare to C. White arrows in B and C indicate lower bacterial accumulation, likely because of a lower amount of motile cells at 12 h (compared with the same area at 6 h). (Scale bars: 200 µm.)

Fig. S3.

B. subtilis cells cannot grow in plant media. Growth curve of B. subtilis cells growing in a 96-well plate in Lysogeny broth bacterial media (LB; red line) and a plant media: Murashige and Skoog (MS; blue line). Values are normalized means of bacterial density measured at OD₆₀₀ (n = 5; independent inoculations for each treatment). Bars indicate ±SD of five independent replicates.

Fig. 3.

B. subtilis is attracted to the root elongation zone. Single images extracted from a time-lapse image series of six Arabidopsis strains with a constitutive GFP reporter (green) marking different root cell types. Each strain was incubated with fluorescently labeled (mKate) B. subtilis cells (red). GFP labeling is in the following root cell types: root hairs (COBL9), cortex (CORTEX), nonhair epidermis (WER), endodermis-quiescent center (SCR), vasculature (WOL), and columella (PET111). The presented images were obtained 2 h after introducing labeled B. subtilis into the TRIS device. The two horizontal dashed lines in the merge row denote the region of primary bacterial colonization. The white arrow in the PET111 GFP image points to the green fluorescent signal coming from the columella cell type. One set of images was presented of three independent experiments for each reporter line. (Scale bars: 200 µm.)

Fig. S4.

Bacterial distribution between different parts of Arabidopsis root. Quantification of bacteria at different parts of the Arabidopsis root. (A) Normalized B. subtilis intensity (red lines) and cobl9 cell layer marker normalized intensity (green lines) as a function of root length over 12 h after bacterial inoculation. Small blue lines mark the borders between the elongation zone and the mature zone at different time points. (B) Normalized bacterial density (bacterial intensity per root length) ratio between the young part and the mature part of the root, indicating specific bacterial colonization to the root tip zone up to 5–6 h postinoculation. The cyan solid line is a fitted line showing ratio change pattern. The presented image is a cobl9 root cell layer captured at 2 h after bacterial inoculation. (Scale bar: 200 µm.)

Fig. 4.

Bacterial competition for root surface colonization. Real-time imaging of two bacterial strains at the root zone. (A) WT Arabidopsis root (bright field) inoculated with E. coli cells (GFP) and imaged every 30 min for 12 h. Images are representative of five independent inoculation experiments; Movie S9 shows the complete experiment. (B) WT Arabidopsis root (bright field) coincubated with a mixture of GFP-labeled E. coli (green) and mKate-labeled B. subtilis (red). White dashed lines in the GFP images delineate the exclusion zone of the E. coli from the root. Images are representative of four independent coinoculation experiments. (C) Quantification of normalized bacterial fluorescence intensity in B at each time point of B. subtilis (red–yellow surface) and E. coli (green–blue surface) as a function of the distance from the root surface over 12 h. (Scale bars: 200 µm.)

Fig. S5.

Quantification of coinoculation of bacterial fluorescence intensity. Bacterial density as a function of time and distance from root surface: B. subtilis (red–yellow surface) and Escherichia coli (green–blue surface). 2D graphs show bacterial density as a function of distance from root surface at 0, 6, and 12 h postinoculation.

Fig. S6.

Bacterial choice assays in the root environment using the double-channel TRIS device. Time-lapse confocal imaging of two Arabidopsis roots growing in parallel in a double TRIS microfluidic channel separated by a semipermeable divider (dashed bars in bright-field images) (Fig. S1) inculcated with fluorescent B. subtilis cells (mKate). (A) Two WT roots (wt.:wt.) incubated with B. subtilis, (B) WT root (wt.; left) and root of a hairless mutant (cpc try; right), and (C) WT root (wt.; left) and root of a hairy mutant (wer myb23; right). The white arrow in wt.:cpc try in B points to a significant increase in the bacterial “cloud” size at the hairless mutant root. Images are representative of three independent inoculation experiments for each group. (Scale bars: 200 µm.) (D–F) Normalized bacterial intensity at the root elongation zone of each root in three groups: (D) wt.:wt., (E) wt.:cpc try, and (F) wt.:wer myb23. Left and right roots’ intensities are indicated as blue and red lines, respectively. Circles are real data points; solid lines are fitted, and dotted lines are boundaries at 95% confidence intervals.

Fig. S7.

TRIS device enables single-root exudate metabolomics. Selected ion chromatogram of a representative metabolite profile of single-root exudates collected from a 10 d WT Arabidopsis root growing in the TRIS device and analyzed by GC coupled to TOF MS. Numbers indicate selected metabolites identified based on authentic standards: 1, phosphoric acid; 2, isoleucine; 3, glycine; 4, succinic acid; 5, pyroglutamic acid; and 6, myoinositol.

Fig. S8.

Generating focused images using the EDF procedure. (A) Raw images of WT Arabidopsis roots (bright field) incubated with red fluorescent-labeled B. subtilis cells (mKate) at three z heights (−40, 0, and +40 µm). (B) MIP and (C) EDF outputs of the raw images shown in A. (D) Binary map used to generate the EDF images in C colored according to three z heights. Shown images are 3 h postinoculation. (Scale bars: 200 µm.)

Fig. S9.

Quantifying bacterial intensity on the root surface. An example of bacterial fluorescence quantification as a function of distance from the root surface. (A) Bright-field image of WT Arabidopsis root. (B) Automated root edges selection (blue line) highlighting the root surface boundaries. (C) Binary mask of the root object. (D) Computer-generated distance map mask layers from the root surface, with a final thickness of 10 µm per layer. Shown roots are 10 d old. (Scale bars: 200 µm.)