A MADS box protein interacts with a mating-type protein and is required for fruiting body development in the homothallic ascomycete Sordaria macrospora

Abstract

MADS box transcription factors control diverse developmental processes in plants, metazoans, and fungi. To analyze the involvement of MADS box proteins in fruiting body development of filamentous ascomycetes, we isolated the mcm1 gene from the homothallic ascomycete Sordaria macrospora, which encodes a putative homologue of the Saccharomyces cerevisiae MADS box protein Mcm1p. Deletion of the S. macrospora mcm1 gene resulted in reduced biomass, increased hyphal branching, and reduced hyphal compartment length during vegetative growth. Furthermore, the S. macrospora Deltamcm1 strain was unable to produce fruiting bodies or ascospores during sexual development. A yeast two-hybrid analysis in conjugation with in vitro analyses demonstrated that the S. macrospora MCM1 protein can interact with the putative transcription factor SMTA-1, encoded by the S. macrospora mating-type locus. These results suggest that the S. macrospora MCM1 protein is involved in the transcriptional regulation of mating-type-specific genes as well as in fruiting body development.

FIG. 1.

Comparative genetic map of mating-type loci from the heterothallic ascomycete S. cerevisiae and the filamentous ascomycetes N. crassa and S. macrospora. The arrowed boxes represent the orientations and sizes of the ORFs in the mating-type loci. Black arrows with white bars indicate genes encoding proteins of unknown function; dark arrows indicate genes encoding homeodomain proteins (HD); white arrows indicate genes encoding α domain proteins (α); and striped arrows indicate genes encoding high-mobility-group domain (HMG) proteins.

FIG. 2.

Nucleotide and deduced amino acid sequences from the S. macrospora mcm1 gene and its flanking regions and alignments with related proteins. (A) Nucleotide and deduced amino acid sequences of the S. macrospora mcm1 gene. Intron sequences are indicated with lowercase letters, and intron consensus sequences are underlined. The MADS box domain is indicated in gray. Putative CAAT box motifs are doubly underlined. A TATA box sequence is boxed in black, and the putative transcriptional start signal is marked in bold italics. A typical polyadenylation signal identified at positions 1005 to 1016 is boxed. (B) MADS box domains of S. macrospora MCM1 (SmMCM1), Mcm1p of S. cerevisiae (ScMCM1, accession no. CAA88409.1), human SRF (HsSRF, CAI13785), Arabidopsis thaliana AGAMOUS (AtAG, P17839), and Antirrhinum majus DEFICIENS (AmDEF, CAA44629), aligned to maximize similarities. Identical amino acid residues are shaded in black, and functionally similar residues are boxed in gray. (C) Alignment of SAM domains of S. macrospora MCM1 (SmMCM1), yeast Mcm1p and Arg80p (ScMCM1 and ScArg80, NP_013756.1), and human SRF (HsSRF).

FIG. 3.

Transcript analysis of S. macrospora wild type. Total RNA and mRNAs were isolated during different developmental stages of the wild type (3 to 7 days). The Northern blot was probed using an mcm1-specific probe. As a control, the blot was striped and reprobed with a gpd-specific probe.

FIG. 4.

Construction of Δmcm1 strain. (A) Schematic illustration of the genomic region of the S. macrospora mcm1 gene and its flanking sequences and generation of the Δmcm1 replacement vector. The arrow represents the mcm1 coding region interrupted by two introns (black boxes). The positions of primers used to amplify the disruption construct from plasmid pMCM1-KO and to verify homologous recombination at the mcm1 locus are indicated. PtrpC, A. nidulans trpC promoter. (B) Southern analysis. The S. macrospora wt strain, primary transformants T1-3 and T1-1, and the T1-3 single-spore isolate S67718 (Δmcm1) were hybridized with the ³²P-labeled probe indicated in panel A. The sizes of the hybridized fragments in wt and knockout transformants are given in panel A. (C) PCR analyses of homologous recombination from the wt and the single-spore isolate S67718 (Δmcm1). Positions of primers and sizes of amplified fragments are indicated in panel A.

FIG. 5.

Vegetative growth of S. macrospora wild-type strain and Δmcm strain. (A) Wild-type S. macrospora hyphae and fruiting bodies after 7 days of growth on cornmeal medium. An mcm1 mutant grown on cornmeal medium formed highly branched and septated mycelia and no mature fruiting bodies. (B) Comparison of wild-type and Δmcm1 mutant hyphae stained with calcofluor white (1 μg/ml). Over a distance of 600 μm, wt hyphae showed seven compartments on average, and in most cases branches could not be observed. The Δmcm1 mutant contained 11 hyphal compartments over a distance of 600 μm, with two branches on average. Bar, 50 μm. (C) Measurement of hyphal compartment length. Measurements were binned into five different compartment lengths, and the number of analyzed compartments was normalized to 100%. Subsequently, compartment lengths were assigned to the segments. Dark and white bars represent compartment lengths of the wt and Δmcm1 strains, respectively.

FIG. 6.

Sexual developmental stages of wild-type and Δmcm1 mutant strains. (A) Strains of S. macrospora were grown on fructification medium for 9 days. Note the complete absence of perithecia in the mutant strain compared with the wt strain. Differential interference contrast microscopy identified ascogonia (wt and Δmcm1), protoperithecia (wt and Δmcm1), young perithecia (only wt strain), perithecia (only wt strain), and ascospores (only wt strain). Strains were grown on fructification medium and examined after growth at 25°C for days, as indicated. Bars represent sizes (μm) as indicated. (B) Distribution of protoperithecia and perithecia produced by the S. macrospora wild type and the Δmcm1 strain. Percentages of small protoperithecia (<60 μm, white bars), protoperithecia (60 to 200 μm, gray bars), and perithecia (>200 μm, black bars) were calculated from 250 fruiting bodies from three different plates.

FIG. 7.

Fluorescence microscopic analysis of Δmcm1 strains expressing mcm1-egfp. The images show a hypha of S. macrospora carrying the chimeric mcm1-egfp gene. (Top) EGFP fluorescence; (middle) Sytox orange staining of nuclei; (bottom) merged image of top and middle panels and the differential interference contrast micrograph.

FIG. 8.

Two-hybrid analyses of MCM1 and mating-type proteins. (A) Localization of MCM1 transcriptional activation domain. The numbers above the ORFs correspond to the lengths of the MCM1 derivatives. Black boxes represent the MADS box domain (M). β-Galactosidase activity was measured in triplicate for each transformant and is reported in units per milligram of protein. (B) Yeast transformants carrying different combinations of pB and pA derivatives (Table 3) were examined for growth on −Leu, −Ura, −His medium plus 20 mM 3-AT (left) or −Leu, −Ura, −Ade medium (right). −, no growth; +, slow growth; ++, growth; +++, strong growth. Measurements of interactions are highlighted in gray. (C) The β-galactosidase activities of the transformants carrying the GAL4-AD and -BD fusion proteins are given in units per milligram of protein.

FIG. 9.

Far-Western analysis of MCM1 homodimerization and MCM1 binding to SMTA-1. CBP-tagged MCM1 (pC-MCM1) and SMTA-1 (pC-SMTA1) fusion proteins, the corresponding inverse fusion constructs (pC-MCMi and pC-SMTAi), and a calmodulin binding protein tag (pCAL-n) were purified, separated by SDS-PAGE, blotted, and overlaid with His-MCM1 fusion protein. Bonded His-MCM1 was detected by the anti-RGS-His-HRP conjugate antibody.