The clock in growing hyphae and their synchronization in Neurospora crassa.

Utilizing a microfluidic chip with serpentine channels, we inoculated the chip with an agar plug with Neurospora crassa mycelium and successfully captured individual hyphae in channels. For the first time, we report the presence of an autonomous clock in hyphae. Fluorescence of a mCherry reporter gene driven by a clock-controlled gene-2 promoter (ccg-2p) was measured simultaneously along hyphae every half an hour for at least 6 days. We entrained single hyphae to light over a wide range of day lengths, including 6,12, 24, and 36 h days. Hyphae tracked in individual serpentine channels were highly synchronized (K = 0.60-0.78). Furthermore, hyphae also displayed temperature compensation properties, where the oscillation period was stable over a physiological range of temperatures from 24 °C to 30 °C (Q10 = 1.00-1.10). A Clock Tube Model developed could mimic hyphal growth observed in the serpentine chip and provides a mechanism for the stable banding patterns seen in race tubes at the macroscopic scale and synchronization through molecules riding the growth wave in the device.

Computer vision models enable mixed linear modeling to predict arbuscular mycorrhizal fungal colonization using fungal morphology.

The presence of Arbuscular Mycorrhizal Fungi (AMF) in vascular land plant roots is one of the most ancient of symbioses supporting nitrogen and phosphorus exchange for photosynthetically derived carbon. Here we provide a multi-scale modeling approach to predict AMF colonization of a worldwide crop from a Recombinant Inbred Line (RIL) population derived from Sorghum bicolor and S. propinquum. The high-throughput phenotyping methods of fungal structures here rely on a Mask Region-based Convolutional Neural Network (Mask R-CNN) in computer vision for pixel-wise fungal structure segmentations and mixed linear models to explore the relations of AMF colonization, root niche, and fungal structure allocation. Models proposed capture over 95% of the variation in AMF colonization as a function of root niche and relative abundance of fungal structures in each plant. Arbuscule allocation is a significant predictor of AMF colonization among sibling plants. Arbuscules and extraradical hyphae implicated in nutrient exchange predict highest AMF colonization in the top root section. Our work demonstrates that deep learning can be used by the community for the high-throughput phenotyping of AMF in plant roots. Mixed linear modeling provides a framework for testing hypotheses about AMF colonization phenotypes as a function of root niche and fungal structure allocations.

The macroscopic limit to synchronization of cellular clocks in single cells of Neurospora crassa.

We determined the macroscopic limit for phase synchronization of cellular clocks in an artificial tissue created by a "big chamber" microfluidic device to be about 150,000 cells or less. The dimensions of the microfluidic chamber allowed us to calculate an upper limit on the radius of a hypothesized quorum sensing signal molecule of 13.05 nm using a diffusion approximation for signal travel within the device. The use of a second microwell microfluidic device allowed the refinement of the macroscopic limit to a cell density of 2166 cells per fixed area of the device for phase synchronization. The measurement of averages over single cell trajectories in the microwell device supported a deterministic quorum sensing model identified by ensemble methods for clock phase synchronization. A strong inference framework was used to test the communication mechanism in phase synchronization of quorum sensing versus cell-to-cell contact, suggesting support for quorum sensing. Further evidence came from showing phase synchronization was density-dependent.