Ines Teichert

The supramolecular striatin-interacting phosphatases and kinases (STRIPAK) complex is highly conserved in eukaryotes and controls diverse developmental processes in fungi. STRIPAK is genetically and physically linked to the Hippo-related septation initiation network (SIN), which signals through a chain of three kinases, including the terminal nuclear Dbf2-related (NDR) family kinase DBF2. Here, we provide evidence for the function of DBF2 during sexual development and vegetative growth of the homothallic ascomycetous model fungus Sordaria macrospora. Using mutants with a deleted dbf2 gene and complemented strains carrying different variants of dbf2, we demonstrate that dbf2 is essential for fruiting body formation, as well as septum formation of vegetative hyphae. Furthermore, we constructed dbf2 mutants carrying phospho-mimetic and phospho-deficient codons for two conserved phosphorylation sites. Growth tests of the phosphorylation mutants showed that coordinated phosphorylation is crucial for controlling vegetative growth rates and maintaining proper septum distances. Finally, we investigated the function of DBF2 by overexpressing the dbf2 gene. The corresponding transformants showed disturbed cytokinesis during ascospore formation. Thus, regulated phosphorylation of DBF2 and precise expression of the dbf2 gene are essential for accurate septation in vegetative hyphae and coordinated cell division during septation and sexual spore formation.

The formation of fruiting bodies is a highly regulated process that requires the coordinated formation of different cell types. By analyzing developmental mutants, many developmental factors have already been identified. Yet, a complete understanding of fruiting body formation is still lacking. In this study, we analyzed developmental mutant pro34 of the filamentous ascomycete Sordaria macrospora. Genome sequencing revealed a deletion in the pro34 gene encoding a putative mitochondrial complex I assembly factor homologous to Neurospora crassa CIA84. We show that PRO34 is required for fast vegetative growth, fruiting body and ascospore formation. The pro34 transcript undergoes adenosine to inosine editing, a process correlated with sexual development in fruiting body-forming ascomycetes. Fluorescence microscopy and western blot analysis showed that PRO34 is a mitochondrial protein, and blue-native PAGE revealed that the pro34 mutant lacks mitochondrial complex I. Inhibitor experiments revealed that pro34 respires via complexes III and IV, but also shows induction of alternative oxidase, a shunt pathway to bypass complexes III and IV. We discuss the hypothesis that alternative oxidase is induced to prevent retrograde electron transport to complex I intermediates, thereby protecting from oxidative stress.

The cloning of plasmids can be time-consuming or expensive. Yet, cloning is a prerequisite for many standard experiments for the functional analysis of genes, including the generation of deletion mutants and the localization of gene products. Here, we provide Golden Gate vectors for fast and easy cloning of gene fusion as well as gene deletion vectors applicable to diverse fungi. In Golden Gate cloning, restriction and ligation occur simultaneously in a one-pot reaction. Our vector set contains recognition sites for the commonly used type IIS restriction endonuclease BsaI. We generated plasmids for C- as well as N-terminal tagging with GFP, mRFP and 3xFLAG. For gene deletion, we provide five different donor vectors for selection marker cassettes. These include standard cassettes for hygromycin B, nourseothricin and phleomycin resistance genes as well as FLP/FRT-based marker recycling cassettes for hygromycin B and nourseothricin resistance genes. To make cloning most feasible, we provide robust protocols, namely (1) an overview of cloning procedures described in this paper, (2) specific Golden Gate reaction protocols and (3) standard primers for cloning and sequencing of plasmids and generation of deletion cassettes by PCR and split-marker PCR. We show that our vector set is applicable for the biotechnologically relevant Penicillium chrysogenum and the developmental model system Sordaria macrospora. We thus expect these vectors to be beneficial for other fungi as well. Finally, the vectors can easily be adapted to organisms beyond the kingdom fungi.

The striatin-interacting phosphatase and kinase (STRIPAK) multi-subunit signaling complex is highly conserved within eukaryotes. In fungi, STRIPAK controls multicellular development, morphogenesis, pathogenicity, and cell-cell recognition, while in humans, certain diseases are related to this signaling complex. To date, phosphorylation and dephosphorylation targets of STRIPAK are still widely unknown in microbial as well as animal systems. Here, we provide an extended global proteome and phosphoproteome study using the wild type as well as STRIPAK single and double deletion mutants (Δpro11, Δpro11Δpro22, Δpp2Ac1Δpro22) from the filamentous fungus Sordaria macrospora. Notably, in the deletion mutants, we identified the differential phosphorylation of 129 proteins, of which 70 phosphorylation sites were previously unknown. Included in the list of STRIPAK targets are eight proteins with RNA recognition motifs (RRMs) including GUL1. Knockout mutants and complemented transformants clearly show that GUL1 affects hyphal growth and sexual development. To assess the role of GUL1 phosphorylation on fungal development, we constructed phospho-mimetic and -deficient mutants of GUL1 residues. While S180 was dephosphorylated in a STRIPAK-dependent manner, S216, and S1343. served as non-regulated phosphorylation sites. While the S1343 mutants were indistinguishable from wild type, phospho-deficiency of S180 and S216 resulted in a drastic reduction in hyphal growth, and phospho-deficiency of S216 also affects sexual fertility. These results thus suggest that differential phosphorylation of GUL1 regulates developmental processes such as fruiting body maturation and hyphal morphogenesis. Moreover, genetic interaction studies provide strong evidence that GUL1 is not an integral subunit of STRIPAK. Finally, fluorescence microcopy revealed that GUL1 co-localizes with endosomal marker proteins and shuttles on endosomes. Here, we provide a new mechanistic model that explains how STRIPAK-dependent and -independent phosphorylation of GUL1 regulates sexual development and asexual growth. In eukaryotes, the striatin-interacting phosphatase and kinase (STRIPAK) multi-subunit signaling complex controls a variety of developmental processes, and the lack of single STRIPAK subunits is associated with severe developmental defects and diseases. However, in humans, animals, as well as fungal microbes, the phosphorylation and dephosphorylation targets of STRIPAK are still largely unknown. The filamentous fungus Sordaria macrospora is a well-established model system used to study the function of STRIPAK, since a collection of STRIPAK mutants is experimentally accessible We previously established an isobaric tag for relative and absolute quantification (iTRAQ)-based proteomic and phosphoproteomic analysis to identify targets of STRIPAK. Here, we investigate mutants that lack one or two STRIPAK subunits. Our analysis resulted in the identification of 129 putative phosphorylation targets of STRIPAK including GUL1, a homolog of the RNA-binding protein Ssd1 from yeast. Using fluorescence microscopy, we demonstrate that GUL1 shuttles on endosomes. We also investigated deletion, phospho-mimetic, and -deletion mutants and revealed that GUL1 regulates sexual and asexual development in a phosphorylation-dependent manner. Collectively, our comprehensive genetic and cellular analysis provides new fundamental insights into the mechanism of how GUL1, as a STRIPAK target, controls multiple cellular functions.

RNA editing occurs in all kingdoms of life and in various RNA species. The editing of nuclear protein-coding transcripts has long been known in metazoans, but was only recently detected in fungi. In contrast to many metazoan species, fungal editing sites occur mostly in coding regions, and therefore, fungal editing can change protein sequences and lead to modified or new functions of proteins. Indeed, mRNA editing is thought to be generally adaptive on fungi. Although RNA editing has been detected in both, Ascomycota and Basidiomycota, there seem to be considerable differences between these two classes of fungi concerning the types, the timing, and the purpose of editing. This review summarizes the characteristics of RNA editing in fungi and compares them to metazoan species and bacteria. In particular, it will review cellular processes affected by editing and speculate on the purpose of editing for fungal biology with a focus on the filamentous ascomycetes. Key Points: • Fungi show various types of mRNA editing in nuclear transcripts. • Fungal editing leads to proteome diversification. • Filamentous ascomycetes may require editing for sexual sporulation. • Wood-degrading basidiomycetes may use editing for adaptation to different substrates.

The striatin-interacting phosphatase and kinase (STRIPAK) multi-subunit signaling complex is highly conserved within eukaryotes. In fungi, STRIPAK controls multicellular development, morphogenesis, pathogenicity, and cell-cell recognition, while in humans, certain diseases are related to this signaling complex. To date, phosphorylation and dephosphorylation targets of STRIPAK are still widely unknown in microbial as well as animal systems. Here, we provide an extended global proteome and phosphoproteome study using the wild type as well as STRIPAK single and double deletion mutants from the filamentous fungus Sordaria macrospora. Notably, in the deletion mutants, we identified the differential phosphorylation of 129 proteins, of which 70 phosphorylation sites were previously unknown. Included in the list of STRIPAK targets are eight proteins with RNA recognition motifs (RRMs) including GUL1. Knockout mutants and complemented transformants clearly show that GUL1 affects hyphal growth and sexual development. To assess the role of GUL1 phosphorylation on fungal development, we constructed phospho-mimetic and -deficient mutants of GUL1 residues S180, S216, and S1343. While the S1343 mutants were indistinguishable from wildtype, phospho-deficiency of S180 and S216 resulted in a drastic reduction in hyphal growth and phospho-deficiency of S216 also affects sexual fertility. These results thus suggest that differential phosphorylation of GUL1 regulates developmental processes such as fruiting body maturation and hyphal morphogenesis. Moreover, genetic interaction studies provide strong evidence that GUL1 is not an integral subunit of STRIPAK. Finally, fluorescence microcopy revealed that GUL1 co-localizes with endosomal marker proteins and shuttles on endosomes. Here, we provide a new mechanistic model that explains how STRIPAK-dependent and - independent phosphorylation of GUL1 regulates sexual development and asexual growth. Author Summary: In eukaryotes, the striatin-interacting phosphatase and kinase (STRIPAK) multi-subunit signaling complex controls a variety of developmental processes, and the lack of single STRIPAK subunits is associated with severe developmental defects and diseases. However, in humans, animals, as well as fungal microbes, the phosphorylation and dephosphorylation targets of STRIPAK are still largely unknown. The filamentous fungus Sordaria macrospora is a well-established model system used to study the function of STRIPAK, since a collection of STRIPAK mutants is experimentally accessible. We previously established an isobaric tag for relative and absolute quantification (iTRAQ)-based proteomic and phosphoproteomic analysis to identify targets of STRIPAK. Here, we investigate mutants that lack one or two STRIPAK subunits. Our analysis resulted in the identification of 129 putative phosphorylation targets of STRIPAK including GUL1, a homolog of the RNA-binding protein SSD1 from yeast. Using fluorescence microscopy, we demonstrate that GUL1 shuttles on endosomes. We also investigated deletion, phospho-mimetic, and -deletion mutants and revealed that GUL1 regulates sexual and asexual development in a phosphorylation-dependent manner. Collectively, our comprehensive genetic and cellular analysis provides new fundamental insights into the mechanism of how GUL1, as a STRIPAK target, controls multiple cellular functions.

Next-generation sequencing techniques have considerably increased the number of completely sequenced eukaryotic genomes. These genomes are mostly automatically annotated, and ab initio gene prediction is commonly combined with homology-based search approaches and often supported by transcriptomic data. The latter in particular improve the prediction of intron splice sites and untranslated regions. However, correct prediction of translation initiation sites (TIS), alternative splice junctions, and protein-coding potential remains challenging. Here, we present an advanced proteogenomics approach, namely, the combination of proteogenomics and de novo peptide sequencing analysis, in conjunction with Blast2GO and phylostratigraphy. Using the model fungus Sordaria macrospora as an example, we provide a comprehensive view of the proteome that not only increases the functional understanding of this multicellular organism at different developmental stages but also immensely enhances the genome annotation quality.

The striatin-interacting phosphatases and kinases (STRIPAK) complex is evolutionary highly conserved and has been structurally and functionally described in diverse lower and higher eukaryotes. In recent years, this complex has been biochemically characterized better and further analyses in different model systems have shown that it is also involved in numerous cellular and developmental processes in eukaryotic organisms. Further recent results have shown that the STRIPAK complex functions as a macromolecular assembly communicating through physical interaction with other conserved signaling protein complexes to constitute larger dynamic protein networks. Here, we will provide a comprehensive and up-to-date overview of the architecture, function and regulation of the STRIPAK complex and discuss key issues and future perspectives, linked with human diseases, which may form the basis of further research endeavors in this area. In particular, the investigation of bi-directional interactions between STRIPAK and other signaling pathways should elucidate upstream regulators and downstream targets as fundamental parts of a complex cellular network.

Fungal fruiting bodies are complex three-dimensional structures that are formed to protect and disperse the sexual spores. Their morphogenesis requires the concerted action of numerous genes; however, at the molecular level, the spatio-temporal sequence of events leading to the mature fruiting body is largely unknown. In previous studies, the transcription factor gene pro44 and the histone chaperone gene asf1 were shown to be essential for fruiting body formation in the ascomycete Sordaria macrospora. Both PRO44 and ASF1 are predicted to act on the regulation of gene expression in the nucleus, and mutants in both genes are blocked at the same stage of development. Thus, we hypothesized that PRO44 and ASF1 might be involved in similar aspects of transcriptional regulation. In this study, we characterized their roles in fruiting body development in more detail. The PRO44 protein forms homodimers, localizes to the nucleus, and is strongly expressed in the outer layers of the developing young fruiting body. Analysis of single and double mutants of asf1 and three other chromatin modifier genes, cac2, crc1, and rtt106, showed that only asf1 is essential for fruiting body formation whereas cac2 and rtt106 might have redundant functions in this process. RNA-seq analysis revealed distinct roles for asf1 and pro44 in sexual development, with asf1 acting as a suppressor of weakly expressed genes during morphogenesis. This is most likely not due to global mislocalization of nucleosomes as micrococcal nuclease-sequencing did not reveal differences in nucleosome spacing and positioning around transcriptional start sites between Δasf1 and the wild type. However, bisulfite sequencing revealed a decrease in DNA methylation in Δasf1, which might be a reason for the observed changes in gene expression. Transcriptome analysis of gene expression in young fruiting bodies showed that pro44 is required for correct expression of genes involved in extracellular metabolism. Deletion of the putative transcription factor gene asm2, which is downregulated in young fruiting bodies of Δpro44, results in defects during ascospore maturation. In summary, the results indicate distinct roles for the transcription factor PRO44 and the histone chaperone ASF1 in the regulation of sexual development in fungi.