Visualization of microbial communities within single leaves

Knowledge of the ecological forces that structure microbial communities is pivotal to designing strategies that maintain diversity and promote greater host fitness. One major force is interspecific bacterial interactions such as cooperation, competition, and predation. These interactions clearly depend on the spatial configuration of community members, which is lost or confounded in most 16S rRNA gene amplicon and shotgun metagenomic surveys.

Here, using the endophytic leaf bacterial community of Arabidopsis thaliana as a model, we visualize the spatial structure of multi-species, in planta bacterial communities, and examine the role of space in the maintenance of diversity. For this study, we take advantage of cultivatable species isolated from A. thaliana. These isolates are among the most abundant species found within the leaves in nature.

We inoculate controlled assemblages of these species into gnotobiotic plants, fix leaves after communities stabilize, and subject sectioned leaf samples to fluorescence in situ hybridization (FISH). Standard bandpass filter imaging limits analysis to only a few fluorophores and becomes convoluted in the presence of strong plant autofluorescence. Therefore, we use spectral imaging to subtract the autofluorescence spectrum and to distinguish overlapping emission spectra. To first confirm the effectiveness of this technique, we imaged mono-colonized leaves to observe each species’ niche preferences in the endophytic environment.
We next include pairwise combinations of isolates and observe how each species modulates its spatial distribution in response to another species. We have preliminary evidence that the plant environment does indeed modulate interspecific bacterial interactions.

Two isolates (Pseudomonas fluorescens and P. poae) that exhibit competitive exclusion in a spatially homogenous liquid media environment, coexist to equal numbers when inoculated into gnotobiotic plants, as determined by spread plating plant homogenates and counting colony forming units. The spatial structure of this community is currently under investigation. By working up from mono-colonized leaves to more complex communities, we demonstrate that the host environment promotes greater diversity by providing for complex spatial distributions. Knowledge of how bacteria spatially distribute in this host environment will inform future strategies for maintaining diversity and host health.