Functional characterization of MAT1-1-specific mating-type genes in the homothallic ascomycete Sordaria macrospora provides new insights into essential and nonessential sexual regulators

Abstract

Mating-type genes in fungi encode regulators of mating and sexual development. Heterothallic ascomycete species require different sets of mating-type genes to control nonself-recognition and mating of compatible partners of different mating types. Homothallic (self-fertile) species also carry mating-type genes in their genome that are essential for sexual development. To analyze the molecular basis of homothallism and the role of mating-type genes during fruiting-body development, we deleted each of the three genes, SmtA-1 (MAT1-1-1), SmtA-2 (MAT1-1-2), and SmtA-3 (MAT1-1-3), contained in the MAT1-1 part of the mating-type locus of the homothallic ascomycete species Sordaria macrospora. Phenotypic analysis of deletion mutants revealed that the PPF domain protein-encoding gene SmtA-2 is essential for sexual reproduction, whereas the alpha domain protein-encoding genes SmtA-1 and SmtA-3 play no role in fruiting-body development. By means of cross-species microarray analysis using Neurospora crassa oligonucleotide microarrays hybridized with S. macrospora targets and quantitative real-time PCR, we identified genes expressed under the control of SmtA-1 and SmtA-2. Both genes are involved in the regulation of gene expression, including that of pheromone genes.

Fig. 1.

Schematic comparison of mating-type loci from heterothallic and homothallic fungi. The arrowed boxes represent the orientation and size of the ORFs in the mating-type locus. Species-specific names and standard nomenclature are given. Known functional domains of the encoded mating-type proteins are given, and genes containing the same functional domain are the same color. α, α domain; HMG, HMG domain; Homeo, homeodomain; PPF, PPF domain; ?, no known domain. Genes essential for sexual development are indicated with an asterisk. Superscripts: 1, homothallic A. nidulans carries two unlinked counterparts of MAT1-1-1 and MAT1-2-1; 2, the homothallic Neurospora species N. africana, N. dodgei, N. galapagosensis, and N. lineolata contain only MAT1-1-specific genes.

Fig. 2.

Sexual development of the WT and deletion mutants ΔSmtA-1, ΔSmtA-2, ΔSmtA-2/3, and ΔSmtA-3. Note the complete absence of perithecia in ΔSmtA-2 and ΔSmtA-2/3 and the WT-like development of ΔSmtA-2 carrying an ectopically integrated SmtA-2 copy. Strains were grown on SWG at 24°C, and pictures of the sexual structures ascogonia, protoperithecia, and perithecia and asci (not ΔSmtA-2 and ΔSmtA-2/3) were taken after 3, 5, and 7 days of growth on SWG.

Fig. 3.

Quantitative real-time PCR analysis of the pheromone precursor genes ppg1 and ppg2 and their cognate receptors pre1and pre2 in ΔSmtA-1 (A), ΔSmtA-2 (B), and ΔSmta-1 (C) compared to the WT. Ratios are given as logarithmic values (base 2) and are means of three independent experiments. Means were calculated with REST (58). The pheromone genes ppg1 and ppg2 in ΔSmtA-1 and ppg2 in ΔSmtA-2 (dark bars) are significantly regulated (P values of 0.001, 0.008, and 0.001, respectively, determined with REST [58]). Shown are averages of three independent experiments ± the standard deviation.

Fig. 4.

Cross-species microarray analysis. (A) Numbers of genes that were found in cross-species microarray experiments to be up- or downregulated in ΔSmtA-1 and ΔSmtA-2, respectively. Genes that were found to be regulated the same way in both mutants compared to the WT are dark gray; genes that are regulated solely in one mutant are light gray. (B) Comparison of 60 genes that are the most strongly regulated in at least one mutant. According to the diagram, 30 are regulated the same way in both mutants (I), 24 are regulated exclusively in one mutant (II), and 2 are regulated the opposite way (III). Analysis was done with MultiExperimentViewer (MeV) (69).

Fig. 5.

Predicted model of the regulation of sexual development and pheromone gene expression in S. macrospora by mating-type proteins. The HMG domain protein SMTa-1 is essential for the pairing of nuclei prior to the formation of dikaryotic ascogenous hyphae and induces a developmental arrest which is overcome by the PPF domain protein SMTA-2. SMTA-3 and SMTA-1 are not involved in this process. However, the α domain protein SMTA-1 retains some function in the regulation of pheromone precursor genes. Positive regulation is shown as an arrow, and negative regulation is shown as a bar.