Chytrid rhizoid morphogenesis resembles hyphal development in multicellular fungi and is adaptive to resource availability

Key to the ecological prominence of fungi is their distinctive cell biology, our understanding of which has been principally based on dikaryan hyphal and yeast forms. The early-diverging Chytridiomycota (chytrids) are ecologically important and a significant component of fungal diversity, yet their cell biology remains poorly understood. Unlike dikaryan hyphae, chytrids typically attach to substrates and feed osmotrophically via anucleate rhizoids. The evolution of fungal hyphae appears to have occurred from rhizoid-bearing lineages and it has been hypothesized that a rhizoid-like structure was the precursor to multicellular hyphae. Here, we show in a unicellular chytrid, Rhizoclosmatium globosum, that rhizoid development exhibits striking similarities with dikaryan hyphae and is adaptive to resource availability. Rhizoid morphogenesis exhibits analogous patterns to hyphal growth and is controlled by β-glucan-dependent cell wall synthesis and actin polymerization. Chytrid rhizoids growing from individual cells also demonstrate adaptive morphological plasticity in response to resource availability, developing a searching phenotype when carbon starved and spatial differentiation when interacting with particulate organic matter. We demonstrate that the adaptive cell biology and associated developmental plasticity considered characteristic of hyphal fungi are shared more widely across the Kingdom Fungi and therefore could be conserved from their most recent common ancestor.


Culture maintenance. Rhizoclosmatium globosum JEL800 was maintained on
PmTG agar plates (Barr, 1986) at 22 °C in the dark. To harvest zoospores for experiments, plates were flooded with 1 ml dH2O and the suspension passed through a 10 μm cell sieve (pluriSelect). Zoospore density was quantified using a Sedgewick Raft Counter (Pyser SCGI) and a Leica DM1000 (10 x objective) with cells fixed in 2% formaldehyde. Zoospores were diluted to a working density of 6.6 x 10 3 ml -1 for all experiments. All experiments detailed below were conducted in Bold's Basal Medium (BBM) supplemented with 1.89 mM ammonium sulfate and 500 µl.l -1 F/2 vitamin solution (Guillard & Ryther, 1967). individual rhizoid compartments extend) were quantified by free-hand tracing and measurement of extending rhizoid compartments (ten rhizoids for each biological replicate, n = 5) separated by a 30 min interval on maximum intensity projected zstacks in Fiji. Only rhizoids growing along 2D axes were included to make these results comparable with hyphal studies. To make data comparable for comparison with hyphal fungi, extension rates were also scaled by rhizoid or hyphal diameter from data published by López-Franco et al (1994). Rhizoid diameter for R. globosum was 0.23 µm, quantified as the mean measurement of 25 separate rhizoid TEM images.
Rhizoid tracing and reconstruction. Z-stacks of rhizoids were imported into the neuron reconstruction software NeuronStudio (Rodriguez et al., 2006, Rodriguez et al., 2008. Rhizoids were semi-automatically traced with the 'Build Neurite' function using the basal point of the sporangium as the rhizoidal origin. Cells grown for 24 h in BBM 10 mM NAG or on chitin beads were too dense to be manually curated and therefore were automatically traced using dynamic thresholding with a minimum neurite length of 2 µm, although due to their high-density tracings should be considered imperfect. For 4D image stacks, the rhizoid was reconstructed in 3D at each 30 min interval. For particle associated and non-associated rhizoids, traced rhizoid systems from individual cells were manually split into their respective categories. Rhizoids were exported as SWC file extensions (Stockley et al., 1993)  Chemical inhibition of rhizoid growth. Autoclaved glass coverslips (VWR) were placed in a culture dish and submerged in 3 ml BBM with 10 mM NAG. Following 1 h of incubation to allow normal zoospore settlement and germination, 1 ml of growth medium was removed from the dish and 1 ml of poison-containing medium was introduced. Caspofungin diacetate (working concentration 1-50 µM) was used to inhibit cell wall β-glucan synthesis and cytochalasin B (working concentration 0.1-10 µM) was used to inhibit actin filament formation. Cells were further incubated for 6 h, which was found to be sufficient to observe phenotypic variation before being removed from the incubator and held at 4 °C prior to imaging. Coverslips were removed from the dishes using EtOH-cleaned forceps and placed cell-side down into a glass bottom dish containing 100 µl of membrane dye.
β-glucan quantification. R. globosum was grown to 250 ml in BBM with 10 mM NAG (n = 5) for 7 d before harvesting by centrifugation at 4,700 rpm for 10 min in 50 ml aliquots and washed in 50 ml MilliQ H2O. The cell pellet from each flask was processed for β-glucans in duplicate using a commercial β-Glucan assay (Yeast & Mushroom) (K-YBGL, Megazyme) following the manufacturer's protocol. A sample of shop-bought baker's yeast was used as a positive control. Glucans were quantified spectrophotometrically using a CLARIOstar® Plus microplate reader (BMG Labtech).
Carbon starvation and growth on chitin beads. To quantify differential rhizoidal growth under carbon replete and carbon deplete conditions, coverslips were placed in a culture dish and submerged in 3 ml growth medium (either carbon-free BBM or BBM with 10 mM NAG). Dishes were then inoculated with zoospores and incubated for either 1, 4, 7 or 24 h, with the 24 h cell z-stacks stitched as described in the fractal analysis. For both sets of experiments, cells were imaged as per the chemical inhibition experiments above.
Chitin beads (New England Biolabs) were washed three times in carbon-free BBM using a magnetic Eppendorf rack and suspended in carbon-free BBM at a working concentration of 1:1,000 stock concentration. Glass bottom dishes containing 3 ml of the diluted beads were inoculated with zoospores and incubated for either 1, 4, 7 or 24 h prior to imaging. For imaging, the culture medium was aspirated off and beads were submerged in 100 µl FM® 1-43. To understand rhizoid development in a starved cell that had encountered a chitin bead, we imaged cells that contacted a chitin bead following development along the glass bottom of the dish.
Statistical Analysis. The comparison between apical and lateral branching was conducted using a Wilcoxon Rank Sum test. Univariate differences in rhizoid morphometrics between experimental treatments were evaluated using Welch's ttests unless stated otherwise. Shapiro-Wilk and Levene's tests were used to assess normality and homogeneity of variance respectively. If these assumptions could not be met, then Wilcoxon Rank Sum was used as a nonparametric alternative. Univariate morphometric differences between particle-associated and nonassociated rhizoids were evaluated using a paired t-test. All data were analysed in RStudio v1.1.456. (R-Studio Team, 2015).