pH Distribution along Growing Fungal Hyphae at Microscale

Abstract

Creating unique microenvironments, hyphal surfaces and their surroundings allow for spatially distinct microbial interactions and functions at the microscale. Using a microfluidic system and pH-sensitive whole-cell bioreporters (Synechocystis sp. PCC6803) attached to hyphae, we spatially resolved the pH along surfaces of growing hyphae of the basidiomycete Coprinopsis cinerea. Time-lapse microscopy analysis of ratiometric fluorescence signals of >2400 individual bioreporters revealed an overall pH drop from 6.3 ± 0.4 (n = 2441) to 5.0 ± 0.3 (n = 2497) within 7 h after pH bioreporter loading to hyphal surfaces. The pH along hyphal surfaces varied significantly (p < 0.05), with pH at hyphal tips being on average ~0.8 pH units lower than at more mature hyphal parts near the entrance of the microfluidic observation chamber. Our data represent the first dynamic in vitro analysis of surface pH along growing hyphae at the micrometre scale. Such knowledge may improve our understanding of spatial, pH-dependent hyphal processes, such as the degradation of organic matter or mineral weathering.

Keywords: Coprinopsis cinerea; bioreporter; hyphosphere; microfluidics; mycosphere; single cell.

Figure 1

Photograph and schematic of the microcosm for in vitro time-resolved pH monitoring at hyphal surfaces. (a) Photograph of the experimental setup consisting of a fungal inoculum placed ca. 1 mm from the lateral opening of the microfluidic device and tubing used to load the bioreporter into to microchannels via the device inlet. (b) Schematic of the microcosm depicted in (a) consisting of an agar patch and microchannels embodied in a PDMS stab. The microchannels allow for the development of a hyphal monolayer in the observation chamber and subsequent loading of the pH bioreporters via an inlet and outlet system. (c) Micrograph showing typical distribution and attachment of Synechocystis sp. PCC6803_peripHlu pH bioreporters (pink dots) along hyphae of C. cinerea in the observation chamber.

Figure 2

Micrographs depicting hyphal development of C. cinerea in the observation chamber and corresponding changes in the pseudo-colours of hyphal-bound Synechocystis sp. PCC6803_peripHlu pH bioreporter cells. The pseudo-colours refer to the overlay of two pH-dependent emission signals (R_I475/I395). (a) Hyphal development at t = 18 h and most pH bioreporters show a pseudo-colour of magenta (pH ~ 6.3). (b) Hyphal development at t = 25 h and most pH bioreporters show a pseudo-colour of red (pH ~ 5.0). For better visibility, the contours of microchannels are marked by white lines.

Figure 3

Time-dependent average pH and histogram of pH distribution on hyphal surfaces of C. cinerea. The pH was assessed by the hyphal-bound Synechocystis sp. PCC6803 bioreporter cells. (a) Bioreporter data encompass the average and standard deviation of n > 2400 cells (circles, (a)). Experiments in the absence of C. cinerea served as controls (triangles; n > 2200 cells). Time was denoted as t = 0 h when the first hyphal tips appeared in the microfluidic observation chamber. The bioreporter cells were loaded to the hyphae at t = 18 h of the noted time. (b) Corresponding pH distribution on hyphal surfaces of C. cinerea at t = 18–25 h.

Figure 4

Longitudinal distribution of sensed pH by Synechocystis sp. PCC6803 bioreporter cells attached to C. cinerea hyphae. Data reflect average pH and pH distribution of n > 2400 cells incubated at various distances along the observation chamber at t = 18 h and t = 25 h. Average data include pH signals from all cells at ±500 µm from given distances. Significantly (p < 0.05) lower pH (≈0.8 pH) was observed near the hyphal tips (cf. at >3000 µm at t = 18 h, and at >4500 µm at t = 25 h) than those at the more mature part at the entrance of the observation chamber (x = 0–500 µm).