Cell Differentiation during Sexual Development of the Fungus Sordaria macrospora Requires ATP Citrate Lyase Activity

Abstract

During sexual development, mycelial cells from most filamentous fungi differentiate into typical fruiting bodies. Here, we describe the isolation and characterization of the Sordaria macrospora developmental mutant per5, which exhibits a sterile phenotype with defects in fruiting body maturation. Cytological investigations revealed that the mutant strain forms only ascus precursors without any mature spores. Using an indexed cosmid library, we were able to complement the mutant to fertility by DNA-mediated transformation. A single cosmid clone, carrying a 3.5-kb region able to complement the mutant phenotype, has been identified. Sequencing of the 3.5-kb region revealed an open reading frame of 2.1 kb interrupted by a 66-bp intron. The predicted polypeptide (674 amino acids) shows significant homology to eukaryotic ATP citrate lyases (ACLs), with 62 to 65% amino acid identity, and the gene was named acl1. The molecular mass of the S. macrospora ACL1 polypeptide is 73 kDa, as was verified by Western blot analysis with a hemagglutinin (HA) epitope-tagged ACL1 polypeptide. Immunological in situ detection of the HA-tagged polypeptide demonstrated that ACL is located within the cytosol. Sequencing of the mutant acl1 gene revealed a 1-nucleotide transition within the coding region, resulting in an amino acid substitution within the predicted polypeptide. Further evidence that ACL1 is essential for fruiting body maturation comes from experiments in which truncated and mutated versions of the acl1 gene were used for transformation. None of these copies was able to reconstitute the fertile phenotype in transformed per5 recipient strains. ACLs are usually involved in the formation of cytosolic acetyl coenzyme A (acetyl-CoA), which is used for the biosynthesis of fatty acids and sterols. Protein extracts from the mutant strain showed a drastic reduction in enzymatic activity compared to values obtained from the wild-type strain. Investigation of the time course of ACL expression suggests that ACL is specifically induced at the beginning of the sexual cycle and produces acetyl-CoA, which most probably is a prerequisite for fruiting body formation during later stages of sexual development. We discuss the contribution of ACL activity to the life cycle of S. macrospora.The fruiting body maturation of filamentous ascomycetes is an attractive model system to study multicellular development in eukaryotes. It involves the formation of the outer structures of the fruiting body but also development of mature ascospores within the fruiting body itself (for reviews, see references 31 and 47). Ascus development starts with the formation of female gametangia called ascogonia. The ascogenous cells are enveloped by sterile hyphae to form fruiting body precursors. Subsequent tissue differentiation gives rise to an outer pigmented peridial tissue, and following caryogamy, inner ascus initials embedded in sterile paraphyses are formed. Mature fruiting bodies from most ascomycetes harbor 200 to 400 asci, which after meiosis and postmeiotic di...

New Insights Into the Roles of NADPH Oxidases in Sexual Development and Ascospore Germination in Sordaria macrospora

“…Strains, media, and growth conditions S. macrospora strains, listed in Table 1, were grown under standard laboratory conditions on complete medium (CM) or cornmeal malt fructification medium (BMM) media (Esser 1982;Nowrousian et al 1999). Cultivation for ascospore germination assays and DNA extraction were performed as described previously (Nowrousian and Cebula 2005;Kamerewerd et al 2008;Teichert et al 2012).…”

Three α-Subunits of Heterotrimeric G Proteins and an Adenylyl Cyclase Have Distinct Roles in Fruiting Body Development in the Homothallic Fungus Sordaria macrospora

“…All S. macrospora strains were cultivated on cornmeal or CM medium (Esser 1982;Nowrousian et al 1999). For supplementation of the Dsac1 strain with cAMP, 3.4 mm of dibutyrylcAMP (db-cAMP) (Biolog Life Science Institute, Bremen, Germany) was added to solid cornmeal medium.…”

“…Growth rates were measured in race tubes as described by Nowrousian and Cebula (2005). Transformation of S. macrospora was performed according to Nowrousian et al (1999) with 0.4 g Glucanex 200 G (Novozymes Switzerland AG, Neumatt, Dittingen, Switzerland) for cell wall degradation. Details for all S. macrospora strains are given in Table 1.…”

“…Moreover, developmental defects are immediately apparent in this self-fertile fungus without the necessity of fertilization (Pö ggeler et al 2006a). To date, several S. macrospora pro mutants that develop protoperithecia but no perithecia have been generated and characterized, thereby revealing essential components of fruiting body development Nowrousian et al 1999Nowrousian et al , 2007aPö ggeler and Kü ck 2004;Kü ck 2005;Engh et al 2007). In addition, despite the fact that S. macrospora completes the sexual cycle without a mating partner, two pheromone-precursor (ppg1 and ppg2) and two pheromonereceptor genes (pre1 and pre2) were shown to be involved in sexual development (Pö ggeler and Kü ck 2000; Mayrhofer and Pö ggeler 2005;Mayrhofer et al 2006;Pö ggeler et al 2006b).…”

pZHK2, a bi-functional transformation vector, suitable for two step gene targeting

“…The resulting plasmid pIG182 (Fig. 2) was used to transform the wild type strain of S. macrospora as described earlier (Nowrousian et al, 1999). For transformation, plasmid pIG182 was linearized, leaving 5.1 and 1.5 kb of homologous sequence at each end of the resistance cassette.…”