Dissecting the root-fungal interface in 3D reveals spatially distinct signalling landscapes

Abstract

Plant rhizospheric interactions represent intricate relationships that determine plant fitness and are crucial for interrogating host-pathogen dynamics, with significant fundamental and translational implications. Most fungal-plant interactions occur in soil - a disordered and granular 3D environment - and hence remain challenging to unravel due to complex regulatory networks. Our current body of evidence characterizing these molecular dialogues largely stems from experimental systems employing soil or in vitro 2D flat plates, hydroponics and gnotobiotic systems. Soil itself features widely varying visco-elasto-plastic material properties, and its inherent opacity precludes direct visualization of the infection progression in complex diseases such as wilts and root rots. Here, we introduce the first such optically transparent, 3D granular growth matrix to recapitulate complex properties of the soil microenvironment, which enables direct, cellular-level visualization of the plant-fungal interface. Our mechanically tunable 3D matrices support long-term co-culture of plants and fungi with compatibility to classical molecular and physiological assays for unravelling the early signalling events and inter-kingdom crosstalk. By leveraging the optical transparency of this matrix, we track fungal development in response to host signals ex-planta with 3D resolution, to report pioneering evidence of hyphal reprogramming preferentially towards the root tips during the early stages of infection. Crucially, we integrate spatiotemporal transcriptomic analyses and discover distinct pathogen-host ex- and in-planta modules during early signalling, which are likely associated with biomimetic soil-like environments. Together, our findings establish an integrable and versatile 3D platform offering an unprecedented view of the pathogen infection processes, which enables fundamental discoveries into the biological regulation of growth and infection. These insights hold immense potential for advancing our understanding of host immune responses and adaptation of filamentous pathogens, as well as open avenues to decipher drought and disease-resistance mechanisms with major agricultural benefits.