Nuclear dynamics in a fungal chimera

Abstract

A fungal colony is a syncytium composed of a branched and interconnected network of cells. Chimerism endows colonies with increased virulence and ability to exploit nutritionally complex substrates. Moreover, chimera formation may be a driver for diversification at the species level by allowing lateral gene transfer between strains that are too distantly related to hybridize sexually. However, the processes by which genomic diversity develops and is maintained within a single colony are little understood. In particular, both theory and experiments show that genetically diverse colonies may be unstable and spontaneously segregate into genetically homogenous sectors. By directly measuring patterns of nuclear movement in the model ascomycete fungus Neurospora crassa, we show that genetic diversity is maintained by complex mixing flows of nuclei at all length scales within the hyphal network. Mathematical modeling and experiments in a morphological mutant reveal some of the exquisite hydraulic engineering necessary to create the mixing flows. In addition to illuminating multinucleate and multigenomic lifestyles, the adaptation of a hyphal network for mixing nuclear material provides a previously unexamined organizing principle for understanding morphological diversity in the more-than-a-million species of filamentous fungi.

Keywords: biological networks; heterokaryon; hydrodynamics.

Fig. 1.

Dynamics of hH1-GFP and hH1-DsRed nuclear populations in a Neurospora crassa chimera. (A) Two homokaryotic mycelia, one with red-labeled nuclei and one with green-labeled nuclei, freely fuse to form a single chimeric colony (see Movie S1 for nuclear dynamics). (Scale bar, 25 μm.) (B) Nucleotypes become more mixed as the colony grows. We measured genetic diversity in 1D colonies (i.e., having a single well-defined growth direction), using the SD of the proportion of hH1-DsRed nuclei between samples of 130 tip nuclei as an index of mixing (Materials and Methods). Lower SDs mean more uniformly mixed nucleotypes. Nucleotypes may not reflect nuclear genotypes because of histone diffusion, so we also measured the mixing index from conidial chains formed after the mycelium had covered the entire 5-cm agar block (red square and dotted line).

Fig. 2.

N. crassa colonies actively mix nuclei introduced up to 16 mm behind the growing tips. (A) (Upper) Transmitted light image of hH1-gfp conidia (circled in green) inoculated into an unlabeled colony. (Scale bar, 1 mm.) (Lower) GFP-labeled nuclei enter and disperse (arrows) through a calcofluor-stained colony. (Scale bar, 20 μm.) Reprinted with permission from Elsevier from ref. . (B) Probability density function (pdf) of dispersed nuclei vs. time after first entry of nuclei into the colony and distance in the direction of growth. Lines give summary statistics: solid line, mean distance traveled by nuclei into colony; dashed line, maximum distance traveled.

Fig. 3.

Rapid dispersal of new nucleotypes is associated with complex nuclear flows. (A) Growing tips at the colony periphery are fed with nuclei from 20–30 mm into the colony interior. Average nuclear speeds are almost identical between wild-type colonies of different ages (key to colors: blue, 3 cm growth; green, 4 cm; red, 5 cm) and between wild-type and so mutant mycelia (orange: so after 3 cm growth). (B) Individual nuclei follow complex paths to the tips (Left, arrows show direction of hyphal flows). (Center) Four seconds of nuclear trajectories from the same region: Line segments give displacements of nuclei over 0.2-s intervals, color coded by velocity in the direction of growth/mean flow. (Right) Subsample of nuclear displacements in a magnified region of this image, along with mean flow direction in each hypha (blue arrows). (C) Flows are driven by spatially coarse pressure gradients. Shown is a schematic of a colony studied under normal growth and then under a reverse pressure gradient. (D) (Upper) Nuclear trajectories in untreated mycelium. (Lower) Trajectories under an applied gradient. (E) pdf of nuclear velocities on linear–linear scale under normal growth (blue) and under osmotic gradient (red). (Inset) pdfs on a log–log scale, showing that after reversal , velocity pdf under osmotic gradient (green) is the same as for normal growth (blue). (Scale bars, 50 μm.)

Fig. 4.

Mathematical models and the hyphal fusion mutant so reveal the separate contributions of hyphal branching and fusion to nuclear mixing. (A) pdf of distance traveled by nuclei entering a so colony. Mean (solid blue) and maximal (dashed blue) dispersal distances are similar to those of wild-type colonies (red curves, reproduced from Fig. 2B). (B) In so colonies, and <3 mm from the tips of a wild-type colony the network is tree-like, with a leading hypha (red arrowhead) feeding multiple tips (green circles). Hyphal flow rate is proportional to the number of tips fed so can be used to infer position in the branching hierarchy. (Inset) correlation of flow rate with number of tips fed in a real hyphal network. Blue, 3-cm colony; green, 4 cm; red, 5 cm . (C) The probability of sibling nuclei being sent to different tips was optimized by Monte Carlo simulations (SI Text). Optimal branching increases from 0.37 in a random branching network (Upper) to a value close to 0.46 (Lower). Branches are color coded by their flow rates. (D) For real colonies the distribution of branches at each stage of the hierarchy (blue, 3-cm mycelium; green, 4 cm; red, 5 cm) is close to optimal (solid black curve and crosses) rather than random branching (dashed black curve). (E) Despite having close to optimal branching, a so chimera becomes unmixed with growth. Conidial chains of a his-3::hH1-gfp; Pccg1-DsRed so + his-3::hH1-gfp; so heterokaryon tend to contain only hH1-GFP so nuclei (Left) or hH1-GFP DsRed so nuclei (Center); compare a heterokaryotic wild-type conidial chain in which hH1-DsRed and hH1-GFP nuclei are evenly mixed (Upper Right). (Scale bars, 20 μm.) Graph showing narrow spread of between wild-type conidial chains (black line) indicates more mixing of nucleotypes than in so (dashed red line).

Fig. 5.

Hyphal velocities are almost uniformly distributed in wild-type mycelia; i.e., fraction of flow carried by a hypha whose speed is v is almost constant up to , independent of colony size (blue, 3-cm mycelium; green, 4 cm; red, 5 cm). We use this result to estimate the variance in travel times for sibling nuclei traveling from the colony interior to a growing hyphal tip (main text).