AMF-SporeChip provides new insights into arbuscular mycorrhizal fungal pre-symbiotic hyphal growth dynamics at the cellular level.

O_LIArbuscular mycorrhizal fungi (AMF) form symbiotic associations with the majority of land plants and deliver a wide range of soil-based ecosystem services. Due to their conspicuous belowground lifestyle in a dark environment surrounded by soil particles, much is still to be learned about the influence of environmental (i.e., physical) cues on spore germination, hyphal morphogenesis and hyphopodium formation in AMF. C_LIO_LITo fill existing gaps in AMF knowledge, we developed a new microfluidic platform - termed the AMF-SporeChip - to immobilise Rhizophagus and Gigaspora spores and confront pre-symbiotic hyphae with physical obstacles. In combination with timelapse microscopy, the fungi could be examined at the cellular level and in real-time. C_LIO_LIThe AMF-SporeChip allowed us to acquire movies with unprecedented visual clarity and therefore identify various exploration strategies of AMF pre-symbiotic hyphae. We witnessed anastomosis formation involving directed hyphal growth in a "stop-and-go" manner, yielding visual evidence of pre-anastomosis signalling and decision-making. Remarkably, we also revealed a so-far undescribed reversible cytoplasmic retraction as part of a highly dynamic space navigation. C_LIO_LIOur findings demonstrated how AMF employ an intricate mechanism of space searching, involving reversible cytoplasmic retraction, branching and directional changes. In turn, the AMF-SporeChip is expected to open many future frontiers for AMF research. C_LI