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. 2017 Feb 8:8:14444.
doi: 10.1038/ncomms14444.

Innovation and constraint leading to complex multicellularity in the Ascomycota

Tu Anh Nguyen  1 , Ousmane H Cissé  2 , Jie Yun Wong  1 , Peng Zheng  1 , David Hewitt  3 , Minou Nowrousian  4 , Jason E Stajich  2 , Gregory Jedd  1
Affiliations

Innovation and constraint leading to complex multicellularity in the Ascomycota

Tu Anh Nguyen et al. Nat Commun. .

Abstract

The advent of complex multicellularity (CM) was a pivotal event in the evolution of animals, plants and fungi. In the fungal Ascomycota, CM is based on hyphal filaments and arose in the Pezizomycotina. The genus Neolecta defines an enigma: phylogenetically placed in a related group containing mostly yeasts, Neolecta nevertheless possesses Pezizomycotina-like CM. Here we sequence the Neolecta irregularis genome and identify CM-associated functions by searching for genes conserved in Neolecta and the Pezizomycotina, which are absent or divergent in budding or fission yeasts. This group of 1,050 genes is enriched for functions related to diverse endomembrane systems and their organization. Remarkably, most show evidence for divergence in both yeasts. Using functional genomics, we identify new genes involved in fungal complexification. Together, these data show that rudimentary multicellularity is deeply rooted in the Ascomycota. Extensive parallel gene divergence during simplification and constraint leading to CM suggest a deterministic process where shared modes of cellular organization select for similarly configured organelle- and transport-related machineries.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Multicellularity and septal pore gating in the fungi.
(a) The phylogeny of representative fungal species with sequenced genomes. CM taxa are shown with grey background. Pe, Pezizomycotina. Ag, Agaricomycotina. Sa, Saccharomycotina. Ta, Taphrinomycotina. N. cra, Neurospora crassa. A. nid, Aspergillus nidulans. T. mel, Tuber melanosporum. S. cer, Saccharomyces cerevisiae. C. alb, Candida albicans. N. irr, Neolecta irregularis. S. com, Saitoella complicata. T. def, Taphrina deformans. S. pom, Schizosaccharomyces pombe. P. gra, Puccinia graminis. S. ros, Sporobolomyces roseus. U. may, Ustilago maydis. L. bic, Laccaria bicolor. C. cin, Coprinopsis cinerea. R. del, Rhizopus delemar. B. den, Batrachochytrium dendrobatidis. (b) A simplified representation of the hypha and pore-associated membranous organelles. The colours of these organelles correspond to the text colours of the group they represent. (c) A Neolecta fruiting body collected from Black Mountain, New Hampshire. Scale bar, 5 mm. (d) Septal pore associated organelles of N. irregularis. The left panel shows an electron dense structure, which is apparently not membrane-delimited. The middle panel shows a septum that has been plugged by a Woronin body-like organelle. The right panel shows a Woronin body-like organelle free in the cytoplasm. The arrows indicate the pore and arrowheads point to the lipid bilayer. Scale bar, 250 nm. This figure is complemented by Supplementary Fig. 1, which shows additional views of the Neolecta fruiting body.
Figure 2
Figure 2. Estimated protein coding capacity of selected fungal genomes.
(a) The estimated number of genes is shown for selected members of the Ascomycota and Basidiomycota. A. fum, Aspergillus fumigatus. A. nig, Aspergillus niger. A. ory, Aspergillus oryzae. B. cin, Botrytis cinerea. C. gla, Candida glabrata. C. gui, Candida guilliermondii. C. lus, Candida lusitaniae. C. glo, Chaetomium globosum. C. imm, Coccidioides immitis. C. neo, Cryptococcus neoformans. D. han, Debaryomyces hansenii. F. gra, Fusarium graminearum. K. lac, Kluyveromyces lactis. M. gri, Magnaporthe grisea. N. hae, Nectria haematococca. P. pas, Pichia pastoris. P. sti, Pichia stipitis. S. cas, Saccharomyces castellii. S. klu, Saccharomyces kluyveri. S. scl, Sclerotinia sclerotiorum. S. nod, Stagonospora nodorum. T. ree, Trichoderma reesei. Y. lip, Yarrowia lipolytica. Other abbreviations are as indicated in the legend of Fig. 1. (b) Gains and losses of gene families at terminal taxa and internal nodes inferred using Dollo parsimony. Branch colour indicates the extent of net gain (green) or net loss (black) as defined in the legend.
Figure 3
Figure 3. Distribution of proteins involved in septal pore gating and hyphal fusion.
(a) Pezizomycotina- and Agaricomycotina-specific proteins associated with septal pore gating are absent in Neolecta. (b) A subset of Pezizomycotina proteins associated with hyphal fusion (HAM-11, HAM-5, HAM-8, SOFT, ADA-1) are not found in Neolecta. Protein sequences are named according to the model system in which they have been most extensively characterized. For aliases, refer to Supplementary Table 6. The PRO-41 homologue in Neolecta irregularis was manually identified by TBLASTN. PRO41 homologues in Batrachochytrium dendrobatidis, Homo sapiens, Monosiga brevicollis, Dictyostelium discoideum were manually added based on published data. Filled squares denote presence, empty squares denote absence. M. bre, Monosiga brevicollis. H. sap, Homo sapiens. D. dis, Dictyostelium discoideum. A. tha, Arabidopsis thaliana. Other abbreviations are as indicated in the legends of Figs 1 and 2.
Figure 4
Figure 4. Distribution of proteins involved in signalling and hyphal morphogenesis.
(a) Genes involved in light- and ROS-related signal transduction are mostly present in Neolecta and other CM taxa, and absent in budding and fission yeasts. (b) The fusion of chitin synthase and myosin motors (CHS-5 and CHS-7) occurred early in the fungal lineage. These sequences were retained in CM taxa and lost in the two yeast lineages. The septal pore associated protein SPA-10 arose before the divergence of Neolecta and Pezizomycotina. Protein sequences are named according to the model system in which they have been most extensively characterized. For aliases, refer to Supplementary Table 6. Filled squares denote presence, empty squares denote absence. (c) Wild type (WT) hyphae make multicellular precursors (arrows), which mature into fruiting bodies upon fertilization (inset, scale bar, 100μm). The spa-10 mutant (Δspa-10) is defective in this developmental pathway. Scale bar, 1 mm.
Figure 5
Figure 5. Conservation and divergence of dynein regulators.
(a) Substitution rate analysis of dynein regulators. For the indicated proteins, the degree of sequence divergence compared with the Pezizomycotina is determined by substitution rate and indicated by the greyscale. A lighter shade indicates greater divergence from the Pezizomycotina. Red text indicates proteins identified by our search for CM-associated sequences. This figure is complemented by Supplementary Fig. 5, which shows substitution rate analysis of dynein complex components. (b) The cartoon depicts the structure the Dynactin complex. Components shown in red were identified as CM-associated. (c) Graphical representation of the p150Glued multiple sequence alignment. On the vertical axis, a positive score represents greater similarity between Neolecta and Pezizomycotina homologues while a negative score represents greater similarity between Neolecta and Saccharomycotina homologues (see Methods). The grey box indicates the range of score that could be generated by chance. The dotted line represents regions missing in yeast sequences. Red background indicates residues conserved in Neolecta and both reference groups. p150Glued domains are indicated on top of the plot based on the structure of mammalian p150Glued (ref. 87). (d) Systematic length variation in p150Glued. * indicates the regions that appear to have undergone significant contraction (Mann–Whitney U-test, P-values<10−3) in the two yeast-containing groups (highlighted with grey background). This figure is complemented by Supplementary Fig. 7a, which shows length distribution of contracted domains.
Figure 6
Figure 6. Conservation and divergence in peroxins.
(a) Substitution rate analysis. For the indicated peroxins, the degree of sequence divergence compared with the Pezizomycotina is determined by substitution rate and indicated by the greyscale. A lighter shade indicates greater divergence from the Pezizomycotina. (b) The cartoon depicts the peroxisomal matrix (left panel) and membrane protein (right panel) import machinery. Red outline indicates proteins identified by the search for CM-associated sequences. The green colour denotes the lipid bilayer and hydrophobic transmembrane domain of PEX-19 substrates. (c) Graphical representation of the PEX-19 multiple sequence alignment. Amphipathic segments mediating PEX-3 binding (α-a) and chaperone activity (α-1) are indicated. (d) Alignment of α-1 segment in members of the Saccharomycotina (black), N. irregularis (blue), and members of the Pezizomycotina (orange). Numbers indicate the position of the last amino acid in the corresponding sequences. Red and blue background indicates residues with negatively and positively charged side chains, respectively. Different shades of green background indicate residue hydrophobicity. (e) Defective peroxisome biogenesis in a Neurospora strain expressing PEX-19 with the α-1 segment from S. cerevisiae. The images show the matrix marker mCherry-PTS1 in the indicated strains. Scale bar, 10 μm. (f) Mean level of cytoplasmic mCherry-PTS1 in the indicated strains. Error bars, s.d. (n=10). Statistical significance of the difference in cytoplasmic mCherry-PTS1 levels is assessed by one-tailed t-test. ** indicates P value <10−3. The inset shows steady-state levels of PEX-19 as determined by western blotting (upper panel). The lower panel of the inset shows coomassie-stained bands, which serve as a loading control.
Figure 7
Figure 7. Novel CM-associated proteins important for hyphal development.
(a) Substitution rate analysis of novel CM-associated genes whose deletion mutants show growth defect. (b) Mean growth rate of wild-type and the indicated deletion strains. Error bars, s.d. (n=5). The significance of growth rate difference between the wild-type and mutants is assessed using one-tailed t-test. * indicates P value <10−2, ** indicates P value <10−3. (c) The localization of the indicated proteins shown by GFP fusion at their native chromosomal loci. The dotted line indicates cell outline. Scale bar, 10 μm. The inset shows the co-localization (merge) of SPZ-1 with the Spitzenkörper marker CHS-1. Inset scale bar, 5 μm. Images in the third column show MIT-1 co-localization with the mitochondrial marker, MitoTracker. (d) Position of predicted transmembrane domains and known domains in the indicated proteins.
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