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

Understanding plant-fungal interactions is crucial for interrogating host-symbiont and host-pathogen dynamics, with significant fundamental and translational implications. Although plants and fungi typically inhabit soil – an opaque, disordered, and granular 3D environment – our current understanding of molecular dialogues largely stems from experimental systems employing 2D flat plates or hydroponic cultures. Here, we introduce a transparent 3D soil-like granular matrix, which enables direct cellular level visualization and long-term interrogation of plant-fungal interactions. We track fungal colonization of plant roots with unprecedented 3D resolution as well as integrate bulk transcriptomic analyses to discover novel gene regulatory programs activated solely under conditions of 3D culture - representing a tranche of unique molecular signatures likely associated with biomimetic soil-like environments. Together, our findings establish a powerful and versatile 3D platform for investigating plant-fungal interactions. These insights hold immense potential for advancing our understanding of plant immune responses and fungal pathogenicity pathways, as well as for the development of drought and disease-resistant cultivars with major agricultural benefits.