Microarray hybridization analysis of light-dependent gene expression in Penicillium chrysogenum identifies bZIP transcription factor PcAtfA.

The fungal velvet complex is a light-dependent master regulator of secondary metabolism and development in the major penicillin producer, Penicillium chrysogenum. However, the light-dependent mechanism is unclear. To identify velvet-dependent transcriptional regulators that show light-regulated expression, we performed microarray hybridizations with RNA isolated from P. chrysogenum ΔPcku70 cultures grown under 13 different long-term, light-dependent growth conditions. We compared these expression data to data from two velvet complex deletion mutants; one lacked a subunit of the velvet complex (ΔPcvelA), and the other lacked a velvet-associated protein (ΔPclaeA). We sought to identify genes that were up-regulated in light, but down-regulated in ΔPcvelA and ΔPclaeA. We identified 148 co-regulated genes that displayed this regulatory pattern. In silico analyses of the co-regulated genes identified six proteins with fungal-specific transcription factor domains. Among these, we selected the bZIP transcription factor, PcAtfA, for functional characterization in deletion and complementation strains. Our data clearly indicates that PcAtfA governs spore germination. This comparative analysis of different microarray hybridization data sets provided results that may be useful for identifying genes for future functional analyses.

Members of the Penicillium chrysogenum velvet complex play functionally opposing roles in the regulation of penicillin biosynthesis and conidiation.

A velvet multisubunit complex was recently detected in the filamentous fungus Penicillium chrysogenum, the major industrial producer of the ß-lactam antibiotic penicillin. Core components of this complex are P. chrysogenum VelA (PcVelA) and PcLaeA, which regulate secondary metabolite production, hyphal morphology, conidiation, and pellet formation. Here we describe the characterization of PcVelB, PcVelC, and PcVosA as novel subunits of this velvet complex. Using yeast two-hybrid analysis and bimolecular fluorescence complementation (BiFC), we demonstrate that all velvet proteins are part of an interaction network. Functional analyses using single- and double-knockout strains clearly indicate that velvet subunits have opposing roles in the regulation of penicillin biosynthesis and light-dependent conidiation. PcVelC, together with PcVelA and PcLaeA, activates penicillin biosynthesis, while PcVelB represses this process. In contrast, PcVelB and PcVosA promote conidiation, while PcVelC has an inhibitory effect. Our genetic analyses further show that light-dependent spore formation depends not only on PcVelA but also on PcVelB and PcVosA. The results provided here contribute to our fundamental understanding of the function of velvet subunits as part of a regulatory network mediating signals responsible for morphology and secondary metabolism and will be instrumental in generating mutants with newly derived properties that are relevant to strain improvement programs.

A homologue of the human STRIPAK complex controls sexual development in fungi.

Sexual development in fungi is a complex process involving the generation of new cell types and tissues - an essential step for all eukaryotic life. The characterization of sterile mutants in the ascomycete Sordaria macrospora has led to a number of proteins involved in sexual development, but a link between these proteins is still missing. Using a combined tandem-affinity purification/mass spectrometry approach, we showed in vivo association of developmental protein PRO22 with PRO11, homologue of mammalian striatin, and SmPP2AA, scaffolding subunit of protein phosphatase 2A. Further experiments extended the protein network to the putative kinase activator SmMOB3, known to be involved in sexual development. Extensive yeast two-hybrid studies allowed us to pinpoint functional domains involved in protein-protein interaction. We show for the first time that a number of already known factors together with new components associate in vivo to form a highly conserved multi-subunit complex. Strikingly, a similar complex has been described in humans, but the function of this so-called striatin interacting phosphatase and kinase (STRIPAK) complex is largely unknown. In S. macrospora, truncation of PRO11 and PRO22 leads to distinct defects in sexual development and cell fusion, indicating a role for the fungal STRIPAK complex in both processes.

Cell-to-cell communication in plants, animals, and fungi: a comparative review.

Cell-to-cell communication is a prerequisite for differentiation and development in multicellular organisms. This communication has to be tightly regulated to ensure that cellular components such as organelles, macromolecules, hormones, or viruses leave the cell in a precisely organized way. During evolution, plants, animals, and fungi have developed similar ways of responding to this biological challenge. For example, in higher plants, plasmodesmata connect adjacent cells and allow communication to regulate differentiation and development. In animals, two main general structures that enable short- and long-range intercellular communication are known, namely gap junctions and tunneling nanotubes, respectively. Finally, filamentous fungi have also developed specialized structures called septal pores that allow intercellular communication via cytoplasmic flow. This review summarizes the underlying mechanisms for intercellular communication in these three eukaryotic groups and discusses its consequences for the regulation of differentiation and developmental processes.

A mutant defective in sexual development produces aseptate ascogonia.

The transition from the vegetative to the sexual cycle in filamentous ascomycetes is initiated with the formation of ascogonia. Here, we describe a novel type of sterile mutant from Sordaria macrospora with a defect in ascogonial septum formation. This mutant, named pro22, produces only small, defective protoperithecia and carries a point mutation in a gene encoding a protein that is highly conserved throughout eukaryotes. Sequence analyses revealed three putative transmembrane domains and a C-terminal domain of unknown function. Live-cell imaging showed that PRO22 is predominantly localized in the dynamic tubular and vesicular vacuolar network of the peripheral colony region close to growing hyphal tips and in ascogonia; it is absent from the large spherical vacuoles in the vegetative hyphae of the subperipheral region of the colony. This points to a specific role of PRO22 in the tubular and vesicular vacuolar network, and the loss of intercalary septation in ascogonia suggests that PRO22 functions during the initiation of sexual development.