Shining a light on the dark world of plant root-microbe interactions.

Interactions between bacteria and roots are critical to the terrestrial ecosystem. The zone of soil immediately surrounding roots is known as the rhizosphere and the surface of the root the rhizoplane (1, 2). This region is of paramount importance to the growth and productivity of plants because it is the main area where they interact with an enormously complex microbial community, the microbiome. Soil may contain up to 10 bacterial species and 10 bacteria per g as well as a diverse archaeal and eukaryotic population (3, 4). Although the diversity of bacteria in the rhizosphere is less than in soil, it is much more active because it is under strong plant selection and has access to root exudation. Plants may release up to 20% of their photosynthate via their roots, which is critical in shaping and possibly farming the root microbiome. This microbiome consists of symbionts, commensals, and pathogens that will interact with roots, the soil, and one another to form a root holobiont that determinesmuch of biological and agricultural productivity. Despite the immense importance of the root environment, it is often overlooked because it is challenging to image and study roots that are underground and are difficult to observe without disturbance. For example, compare the ease of sampling shoots relative to roots. One of the principal ways of overcoming this is to image roots with X-ray computed tomography scanning, a powerful way of building 3D images of root growth in a spatial and temporal fashion (5). However, this approach is not suited to examine the critical interaction of microbes with roots. To overcome this, plants can be grown between thinly separated glass sheets, known as rhizotrons, on agar plates or similar artificial media and imaged by a wide range of physical and microscopic techniques. This tends to be done at one time point and does not allow dynamic mapping of the spatial and temporal interaction ofmicrobeswith roots. A further recent advance is the use of a microfluidics-based RootChip to monitor root development (6). However, a breakthrough in the live imaging of roots and bacteria is described in the paper by Massalha et al. (7) using a technique called TRIS (tracking root interactions system). For TRIS analysis, roots are grown into a narrow root chamber (160 μM high) contained in a microfluidics device. Bacteria can then be introduced into this chamber and imaged with a wide variety of techniques, including dark-field and confocal microscopy. The microfluidics chamber was made by using soft lithography to etch a single sheet of polydimethylsiloxane with nine separate chambers, each of which contains a single Arabidopsis root, with separate input and output channels through which bacteria can be introduced. Bright-field microscopy can be used to image unlabeled bacteria but the technique is most powerful when fluorescently labeled bacteria are added and imaged by confocal Fig. 1. A cartoon of a root is shown where two bacteria, B. subtilis (in red) and E. coli (in blue), are competing to attach to A. thaliana. B. subtilis has been shown by TRIS to accumulate rapidly at the root elongation zone with later accumulation occurring higher up the root. Arrows and question marks highlight what might happen in steps subsequent to accumulation at the root elongation zone because the order of events is not clear, particularly in soil where the root will be occupied by existing bacteria in biofilms. Bacteria in biofilms are shown without flagella to indicate that motility is usually suppressed at the sessile stage of biofilm formation.