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

Arbuscular 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 anastomosis/hyphal healing mechanisms. To fill existing gaps in AMF knowledge, we developed a new microfluidic platform – the AMF-SporeChip – to visualise the foraging behaviour of germinating Rhizophagus and Gigaspora spores and confront asymbiotic hyphae with physical obstacles. In combination with timelapse microscopy, the fungi could be examined at the cellular level and in real-time. The AMF-SporeChip allowed us to acquire movies with unprecedented visual clarity and therefore identify various exploration strategies of AMF asymbiotic hyphae. We witnessed tip-to-tip and tip-to-side hyphal anastomosis formation. Anastomosis involved 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, including the formation of up to 8 septa upon retraction, as part of a highly dynamic space navigation, probably evolved to optimise foraging efficiency. Our 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.


Media prepara)on
For the preparaGon of MSR(-) medium, used for the hyphal compartment of the arbuscular mycorrhizal fungi (AMF) co-culture, 0.59 g Modified Strullu and Romand (MSR, Duchefa Biochemie, the Netherlands) powder was dissolved in 1 l sterile double disGlled water (ddH2O) containing 10 ml of a 0.15 M calcium nitrate soluGon.The pH was adjusted to 5.5 using 0.1 M NaOH and HCl, and finally 3 g phytagel (Merck, Germany) added.MSR medium, used for chicory root culture and the root compartment of the AMF co-culture, was prepared in the same manner, however, 10 g sucrose were added to the soluGon before adjusGng the pH. 1,2AYer autoclaving, the media must be kept at 60 °C to prevent it from solidifying.Once cooled down and solidified it cannot be melted again.For the on-chip experiments, a liquid version of the MSR(-) medium was prepared; the pH was adjusted to 7 and no phytagel was added (the pH of the phytagel-containing media was measured to be 7 before autoclaving).

AMF culture
The AMF strains Rhizophagus irregularis MUCL 43194, Rhizophagus irregularis MUCL 41833 and Rhizophagus intraradices MUCL 49410 were maintained in vitro in bi-compartmented Petri plates (Ø = 90 mm) on Ri T-DNA transformed roots of chicory (Cichorium intybus L.) on MSR medium.One compartment was filled with 15-20 ml of MSR(-) (termed the "hyphal compartment"), such that the liquid medium is held by the central wall due to surface tension.
Once the MSR(-) medium has solidified, 5-15 ml of medium was carefully poured into the second compartment (termed the "root compartment") while GlGng the plate towards the hyphal compartment (i.e., to ensure connecGvity between the two compartments).The root compartment was then inoculated with a fresh and vital chicory root, the plates were then sealed with cling film and incubated at 27 °C in the dark in inverted posiGon for one week.Plugs of medium containing spores from an at least 3-month-old culture plate showing a high spore density were selected and transferred to an empty sterile Petri plate.These plugs were then cut into very thin pieces and mixed with a scalpel.The viscous spore-containing paste was then spread directly onto the chicory roots.The plates were then sealed with cling film and incubated at 27 °C in the dark in inverted posiGon unGl the spores were mature (usually aYer 3-6 months).
Spores of Gigaspora margarita BEG 34 were produced in pot culture in double-autoclaved (121 °C for 15 min) substrate composed of thin quartz (0.4-0.8 mm), larger quartz (1-2 mm) and calcinated clay (2/1/2; v/v/v) with Plantago lanceolata.AYer at least 9 months of culGvaGon, spores were sampled and disinfected as described by Cranenbrouck et al. 2 Briefly, spores were disinfected using a filtraGon apparatus connected to a vacuum outlet.First, they were cleaned twice in sterile water, then treated with chloramine T 2% soluGon (with 2 drops of Tween 20) for 10 min.Spores were then washed again 3 Gmes with sterile water before treatment with a filtered (using a 0.22 µm Acrodisk filter) anGbioGc soluGon composed of streptomycine sulfate 0.02% and gentamycine sulfate 0.01%, for 10 min.Spores were stored in sterile water at 4 °C before use.

Microfluidic device fabrica)on
The device designs were drawn in AutoCAD 2022 (Autodesk) and checked for correct structuring using KLayout. 3The design was then printed to create a mylar® film photolithography mask by Micro Lithography Services Ltd., UK.For the master mould manufacturing (conducted in a clean room), a fresh silicon wafer (Diameter: 4"; OrientaGon: <100>; Dopant: P(Boron); ResisGvity: 0-100 ohm-cmCz; Centre Thickness: 425-550 µm; Surface: Single side polished, Si-Mat Silicon Materials, Germany) was washed with acetone and isopropanol and subsequently plasma-cleaned for 5 min using a plasma cleaner (PDC-002-CE, Harrick Plasma, USA).The following procedure was used to prepare the master mould for Rhizophagus spp.devices, with parameters for the Gi margarita master mould wriqen in parentheses if deviaGng from the standard protocol.For the first layer, 5 ml of SU-8 2010 (Kayaku, USA) were spin-coated onto the wafer, spinning at 500 rpm for 10 s and 3700 rpm for 30 s (Spin Coater WS-650MZ Modular, Laurell, USA), followed by soY baking at 65 °C for 30 s and 95 °C for 2.5 min ramping up with 5 °C/min on a hot plate (Hot Plate EMS 1000-3, Ascon Tecnologic, Italy).In the next step, the wafer was exposed to ultraviolet (UV) light at a dose of 130 mJ/cm 2 using a UV KUB-3 mask aligner (Kloé SA, France) and baked at 65 °C for 30s and 95 °C for 3.5 min, ramping up with 5 °C/min.For the second layer, 5 ml SU-8 2075 (Kayaku, USA) (SU-8 2150 (Kayaku, USA)) were spun onto the first layer at 500 rpm for 20 s and 2200 rpm (1300 rpm) for 30 s, followed by soY baking at 65 °C for 5 min (7 min) and 95 °C for 15 min (60 min) ramping up with 5 °C/min on a hot plate.For a beqer alignment of the first and second layer, the SU-8 2010 was removed from the alignment marks using acetone and clean room sampling swab (Berkshire, UK).The layers were aligned and the wafer exposed with 228 mJ/cm 2 (450 mJ/cm 2 ) UV light using the UV KUB-3 mask aligner and subsequently baked at 65 °C for 3.5 min (5 min) and 95 °C for 9 min (35 min) ramping up with 5 °C/min on a hot plate.For the development step, the wafer was agitated in SU-8 developer (2-(1-methoxy)propyl acetate, 99%, Thermo ScienGfic, USA) for 8 min (16 min), then for 1 min (4 min) with fresh developer and finally washed with isopropanol and dried with an airgun.In the last step, the master mould was hard baked at 150 °C for 2 min (5 min) on a hot plate.

Imaging
To obtain Gme-lapse images (used to produce growth and germinaGon videos) as well as Gmepoint large images (used for measuring growth rates) two inverted microscopes (Eclipse Ti-U and Eclipse Ti-2, Nikon) were employed.The microscopes were equipped with air immersed ×10/0.3NA (numerical aperture) and x20/0.45NA Plan Fluor objecGves (Nikon; used for Gmepoint images and Gme-lapse experiments respecGvely), motorised stages and camera heads (Ti-U: ReGga R1 CCD camera (Qimaging, Canada); Ti-2: DS-Qi2 Mono Digital Microscope Camera (Nikon, UK)).For growth rate experiments, the large image funcGon in NIS-Elements Advanced Research imaging soYware (Nikon) was used to acquire images once every 24 h for 7 days, with a final image being taken aYer 14 days.To cover the enGre device, 9 × 5 fields of view were needed; the acquired image array was then automaGcally sGtched together with a 20% overlap.For long term microscopy, a temperature-controlled incubator (Okolab, Italy) mounted around the microscope stage was set to 27 °C.

On-plate experiment
For the on-plate experiment, spores were picked up with a pipeqe (Pipetman G, 200 µl, Gilson, USA) and evenly distributed on a glass-boqomed Petri dish (Ø dish = 50 mm, Ø glass = 40 mm; Fluorodish, World Precision Instruments, Germany) containing a thin layer of MSR(-) (ca.1-2 mm).The plates were then sealed with cling film and incubated at 27 °C.Spore germinaGon and hyphal growth were imaged and measured alongside the microdevices in the same manner.

Supplementary Fig. 3 :
SchemaKc showing details of the open obstacle-type structures, Pac-Man, open-box and restricted open-box (from le` to right) with internal angles and contact interfaces indicated in red.Pac-Man: internal angle = 90°, number of contact interfaces = 2; open box: internal angles = 90°, number of contact interfaces = 3; restricted open-box: internal angles = 67°, number of contact interfaces = 3.