Transcriptome and functional analysis of mating in the basidiomycete Schizophyllum commune

Abstract

In this study, we undertook a functional characterization and transcriptome analysis that enabled a comprehensive study of the mating type loci of the mushroom Schizophyllum commune. Induced expression of both the bar2 receptor and the bap2(2) pheromone gene within 6 to 12 h after mates' contact was demonstrated by quantitative real-time PCR. Similar temporal expression patterns were confirmed for the allelic bbr1 receptor and bbp1 pheromone-encoding genes by Northern hybridization. Interestingly, the fusion of clamp connections to the subterminal cell was delayed in mating interactions in which one of the compatible partners expressed the bar2 receptor with a truncated C terminus. This developmental delay allowed the visualization of a green fluorescent protein (Gfp)-labeled truncated receptor at the cell periphery, consistent with a localization in the plasma membrane of unfused pseudoclamps. This finding does not support hypotheses envisioning a receptor localization to the nuclear membrane facilitating recognition between the two different nuclei present in each dikaryotic cell. Rather, Gfp fluorescence observed in such pseudoclamps indicated a role of receptor-pheromone interaction in clamp fusion. Transcriptome changes associated with mating interactions were analyzed in order to identify a role for pheromone-receptor interactions. We detected a total of 89 genes that were transcriptionally regulated in a mating type locus A-dependent manner, employing a cutoff of 5-fold changes in transcript abundance. Upregulation in cell cycle-related genes and downregulation of genes involved in metabolism were seen with this set of experiments. In contrast, mating type locus B-dependent transcriptome changes were observed in 208 genes, with a specific impact on genes related to cell wall and membrane metabolism, stress response, and the redox status of the cell.

Fig 1

Gene bar2 of Schizophyllum commune. The primers S1 and S2 were used for amplification of full-length gene bar2f (NCBI accession number X91168.4) with promoter region (P). The gene bar2t was truncated at the 3′ PstI restriction site (see also NCBI accession number X91168.2). The 3× HA-eGfp tag (ha-egfp) is indicated at the C terminus of the bar2t gene. The white squares indicate introns.

Fig 2

Diagram of interactions screened to identify genes differentially regulated. The comparison of assumed different (green) and similar (red) interactions was used to identify genes regulated in transcriptome profiling.

Fig 3

(A and B) Expression of the pheromone receptor gene bar2 (A) and the pheromone gene bap2(2) (B) in a wild-type mating (12-43 × 4-39); (C) expression of a truncated version of pheromone receptor gene bar2t in the transformant Vbar2t in a mating with the wild-type strain 4-39. The gene expression levels were determined by quantitative real-time PCR of compatible mating interactions over a time period of 72 h. The expression levels in monokaryons (t₀) were normalized to 1, and all other mating times (t₃ to t₇₂) are shown relative to t₀. (D) Ratio of bar2t/bar2 expression, illustrating the effect of a C-terminal truncation of the pheromone receptor.

Fig 4

Northern hybridization of receptor and pheromone gene expression during compatible and semicompatible matings. (A) Expression of the Bbr1 receptor and the Bbp1 pheromone-encoding genes in a compatible mating, 4-40 (M1; A_4,6;B_1,1) × 4-39 (M2; A_1,1;B_3,2), and the two respective monokaryons, as well as an incompatible cross between identical monokaryons (M1x). RNA extraction was performed after the indicated times (3, 6, 12 h). (B) Expression of the receptor genes encoding Bar1 and BbrI and also those encoding the pheromones Bbp1(1), Bbp1(2), and Bbp1(3). Lane 1, expression in A_on semicompatible mating between strains 23 (A_4,6;B_3,1) × 684 (A_2,6;B_3,1); no signal was expected for bar1 since neither of the B loci encodes the bar1 receptor; lanes 2 and 3, B_on semicompatible matings between strains 1792-114-10 (A_4,6;B_3,6) × 4-40 (A_4,6;B_1,1) and 43/26 (A_4,6;B_3,1) × 4-40 (A_4,6;B_1,1), respectively; lane 4, A_on;B_on fully compatible mating between strains 4-40 (A_4,6;B_1,1) × 4-39 (A_1,1;B_3,2); lane 5, strain 4-39 (A_1,1;B_3,2); no signal was expected in this control since no sequences encoding either the Bar1 and Bbr1 receptor or any of the three Bbp1 pheromones are present in strain 4-39; lane 6, strain 4-40 (A_4,6;B_1,1); lane 7, strain 23 (A_4,6;B_3,1). All strains were grown for 8 h after mating. Each well contains 20 μg of total RNA. Expression of glyceraldehyde-3-phosphate dehydrogenase (GPD) was monitored as a loading control.

Fig 5

Pseudoclamp formation in transformants carrying the truncated pheromone receptor gene bar2t after a compatible mating. (a) Pseudoclamp with a DAPI-stained nucleus trapped in the unfused clamp. Bar, 5 μm. (b) Bright-field micrograph of hyphae and DAPI staining of nuclei showing a nuclear pair in the tip cell and the two nuclei separated in the unfused clamp and subterminal cell (asterisk). The tip of the hypha is at the extreme right of the figure, and nuclei are indicated by an arrow. Bar, 20 μm.

Fig 6

States of clamp fusion in a wild-type (wt) dikaryon (12-43 × 4-39) and in two pheromone receptor transformants carrying either the full-length gene bar2f (Vbar2f) or the truncated version, bar2t (Vbar2t), after interacting with a compatible mating partner (strain 4-39); n = 100.

Fig 7

Fruiting bodies of Schizophyllum commune in the wild type (a and b), pheromone receptor transformant Vbar2t with the truncated receptor (c to e), and the pheromone receptor transformant Vbar2f with the full-length receptor gene (f and g). All strains have been mated with the compatible partner strain 4-39. While the wild type forms fruit bodies with ordinary pseudolamellae, the transformants show defects in fruit body development, resulting in fewer or absent pseudolamellae. Bars, 0.5 cm.

Fig 8

Spores of Schizophyllum commune in the wild type (a and b) or pheromone receptor transformants encoding either the truncated receptor (Vbar2t) (c and d) or the full-length receptor (Vbar2f) (e and f). Spores were obtained from fruiting bodies generated from compatible crosses with strain 4-39. While almost all wild-type spores contained two nuclei (b), more than 70% of the spores from the truncated receptor transformants did not contain nuclei and only mitochondrial DNA was stained (d). The spores derived from outcrosses of the full-length receptor transformant Vbar2f showed a higher incidence of two nuclei than spores derived from outcrosses with the truncated version (f).

Fig 9

(A) Micrographs of HA-Gfp-tagged pheromone receptor localization in dikaryotic S. commune transformants by immunostaining (Aa to Ad) and in vivo (Ae and Af) in dikaryotic mycelium (Vbar2tG1 or Vbar2tG11 mated to 4-39); (B) differentiation of pseudoclamps showing strong receptor staining while others are nonfluorescent (top, bright field; middle, DAPI and calcofluor; bottom, Gfp). Bars, 5 μm.

Fig 10

Transcriptional regulation of genes associated with various mating interactions in S. commune. Functional groups of regulated genes (change, ≥5-fold; P ≤ 0.05; green, upregulation; red, downregulation). KOG classification: cellular processes and signaling (M, cell wall/membrane/envelope biogenesis; N, cell motility; O, posttranslational modification, protein turnover, chaperones; T, signal transduction; U, intracellular trafficking, secretion, and vesicular transport; V, defense mechanisms; W, extracellular structures; Y, nuclear structure; Z, cytoskeleton), information storage and processing (A, RNA processing and modification; B, chromatin structure and dynamics; J, translation, ribosomal structure and biogenesis; K, transcription; L, replication, recombination, and repair), metabolism (C, energy production and conversion; D, cell cycle control, cell division, chromosome partitioning; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; P, inorganic ion transport and metabolism; Q, secondary metabolites biosynthesis, transport and catabolism), and poorly characterized (R, general function prediction only; S, function unknown).