Whole plant-environment microscopy reveals how Bacillus subtilis utilises the soil pore space to colonise plant roots

Plant growth is supported by complex interactions with many biophysical elements of their environment including microorganisms, geochemicals, water and gas, all within the otherwise complex and heterogeneous soils physical environment. Our understanding of plant-environment interactions in soil are limited by the difficulty of observing such interactions at the microscopic scale which occur throughout the large volume of influence of the plant. Here, we present the development of 3D live microscopy approaches for resolving plant-microbe interactions across the environment of an entire seedling root growing in a transparent soil in tailor-made mesocosms, maintaining physical conditions for the culture of both plants and microorganisms. A dual-illumination light-sheet system was used to acquire scattering signals from the plant whilst fluorescence signals were captured from transparent soil particles and labelled microorganisms, allowing the generation of quantitative data on samples approximately 3600 mm3 in size with as good as 5 m resolution at a rate of up to one scan every 30 minutes. The system can track the movement of Bacillus subtilis populations in the rhizosphere of lettuce plants in real time, revealing previously unseen patterns of activity. Motile bacteria favoured small pore spaces over the surface of soil particles, colonising the root in a pulsatile manner. Migrations appeared to be directed first towards the root cap as the point "first contact", before subsequent colonisation of mature epidermis cells. Our findings show that microscopes dedicated to live environmental studies present an invaluable tool to understand life in soils. SignificanceBetter knowledge of microbial movement and interaction with plant roots is essential to understanding soil ecosystems. However, the lack of a suitable approach for observing biological activity in such environments severely impedes advances in this field of research. Here, we overcome this major limitation by combining the use of transparent soil with cutting edge live microscopy techniques. We performed a detailed analysis of the movements of Bacillus subtilis and revealed how the soil pore structure influences the behaviour of the bacteria, both before and during the formation of biofilms on the root surface. This work sheds light on previously unseen phenomenon, and accelerates our understanding of soil dwelling organisms which were, before now, unobserved in their native environment.