The Composition and the Structure of MCC/Eisosomes in Neurospora crassa

MCC/eisosomes are protein-organized domains in the plasma membrane of fungi and algae. However, the composition and function(s) of MCC/eisosomes in the filamentous fungus Neurospora crassa were previously unknown. To identify proteins that localize to MCC/eisosomes in N. crassa, we isolated proteins that co-purified with the core MCC/eisosome protein LSP-1, which was tagged with GFP. Proteins that co-fractionated with LSP-1:GFP were then identified by mass spectrometry. Eighteen proteins were GFP-tagged and used to identify six proteins that highly colocalized with the MCC/eisosome marker LSP-1:RFP, while five other proteins showed partial overlap with MCC/eisosomes. Seven of these proteins showed amino acid sequence homology with proteins known to localize to MCC/eisosomes in the yeast Saccharomyces cerevisiae. However, homologs of three proteins known to localize to MCC/eisosomes in S. cerevisiae (Can1, Pkh1/2, and Fhn1) were not found to colocalize with MCC/eisosome proteins in N. crassa by fluorescence microscopy. Interestingly, one new eisosome protein (glutamine-fructose-6-phosphate aminotransferase, gene ID: NCU07366) was detected in our studies. These findings demonstrate that there are interspecies differences of the protein composition of MCC/eisosomes. To gain further insight, molecular modeling and bioinformatics analysis of the identified proteins were used to propose the organization of MCC/eisosomes in N. crassa. A model will be discussed for how the broad range of functions predicted for the proteins localized to MCC/eisosomes, including cell wall synthesis, response and signaling, transmembrane transport, and actin organization, suggests that MCC/eisosomes act as organizing centers in the plasma membrane

Higher yields of cyclodepsipetides from Scopulariopsis brevicaulis by random mutagenesis

The ascomycete Scopulariopsis brevicaulis, which was isolated from the marine sponge Tethya aurantium, produces two cyclodepsipeptides, scopularides A and B [1]. Both peptides exhibit activity against several tumor cell lines. Within the EU-project MARINE FUNGI (EU FP7, 265926) one of our aims is to enhance the production of these secondary metabolites. We are in the process to establish two ways of random mutagenesis by both UV radiation and transposon-mediated. To this end we created UV-mutants and a miniaturised screening method was developed. UV-radiation was performed at 312 nm and the survival rate was set to 1 %. With this method a mutant library was established. To screen these mutants for higher secondary metabolites production, we developed a miniaturised screening method which includes decreased cultivation volume, fast extraction and an optimised LC-MS analysis format. Using the UV mutagenesis, we were able to identify several mutants with a higher scopularide production in comparison to the wild type. One of these mutants, which produces three times more biomass and more than double the amount of scopularide A, has been used for another round of mutation. Next generation sequencing is being employed to identify the molecular genetic basis of the observed mutations. In parallel we employ transposable elements to introduce mutants [2]. The impact of transposons on gene expression as well as their ability to cause major mutations within the genome or single genes makes them an interesting tool for random mutagenesis [3, 4, 5]. We employ the Vader transposon in its homologous host and found that Vader mostly integrates within or very close to genes. Thus it appears to be a useful tool for transposon-mediated mutagenesis in A. niger (6). At current we try to enhance its usability by modifying the Vader element

Development and Validation of a Fast and Optimized Screening Method for Enhanced Production of Secondary Metabolites Using the Marine Scopulariopsis brevicaulis Strain LF580 Producing Anti-Cancer Active Scopularide A and B

Natural compounds from marine fungi are an excellent source for the discovery and development of new drug leads. The distinct activity profiles of the two cyclodepsipeptides scopularide A and B against cancer cell lines set their marine producer strain Scopulariopsis brevicaulis LF580 into the focus of the EU project MARINE FUNGI. One of the main goals was the development of a sustainable biotechnological production process for these compounds. The secondary metabolite production of strain LF580 was optimized by random mutagenesis employing UV radiation. For a fast and reliable detection of the intracellular secondary metabolite production level, a miniaturized bioactivity-independent screening method was developed, as the random mutagenesis yielded a large number of mutants to be analysed quantitatively and none of the existing hyphenated bioassay-dependent screening systems could be applied. The method includes decreased cultivation volume, a fast extraction procedure as well as an optimized LC-MS analysis. We show that deviation could be specifically reduced at each step of the process: The measuring deviation during the analysis could be minimized to 5% and technical deviation occurring in the downstream part to 10–15%. Biological variation during the cultivation process still has the major influence on the overall variation. However, the approach led to a 10-fold reduction of time and similar effects on costs and effort compared to standard reference screening methods. The method was applied to screen the UV-mutants library of Scopulariopsis brevicaulis LF580. For validation purposes, the occurring variations in the miniaturized scale were compared to those in the classical Erlenmeyer flask scale. This proof of concept was performed using the wild type strain and 23 randomly selected mutant strains. One specific mutant strain with an enhanced production behavior could be obtained

The P-type pentatricopeptide repeat protein DWEORG1 is a non-previously reported rPPR protein of Arabidopsis mitochondria

Gene expression in plant mitochondria is mainly regulated by nuclear-encoded proteins on a post-transcriptional level. Pentatricopeptide repeat (PPR) proteins play a major role by participating in mRNA stability, splicing, RNA editing, and translation initiation. PPR proteins were also shown to be part of the mitochondrial ribosome (rPPR proteins), which may act as regulators of gene expression in plants. In this study, we focus on a mitochondrial-located P-type PPR protein-DWEORG1-from Arabidopsis thaliana. Its abundance in mitochondria is high, and it has a similar expression pattern as rPPR proteins. Mutant dweorg1 plants exhibit a slow-growth phenotype. Using ribosome profiling, a decrease in translation efficiency for cox2, rps4, rpl5, and ccmFN2 was observed in dweorg1 mutants, correlating with a reduced accumulation of the Cox2 protein in these plants. In addition, the mitochondrial rRNA levels are significantly reduced in dweorg1 compared with the wild type. DWEORG1 co-migrates with the ribosomal proteins Rps4 and Rpl16 in sucrose gradients, suggesting an association of DWEORG1 with the mitoribosome. Collectively, this data suggests that DWEORG1 encodes a novel rPPR protein that is needed for the translation of cox2, rps4, rpl5, and ccmFN2 and provides a stabilizing function for mitochondrial ribosomes

The Genome and Development-Dependent Transcriptomes of Pyronema confluens: A Window into Fungal Evolution

Fungi are a large group of eukaryotes found in nearly all ecosystems. More than 250 fungal genomes have already been sequenced, greatly improving our understanding of fungal evolution, physiology, and development. However, for the Pezizomycetes, an early-diverging lineage of filamentous ascomycetes, there is so far only one genome available, namely that of the black truffle, Tuber melanosporum, a mycorrhizal species with unusual subterranean fruiting bodies. To help close the sequence gap among basal filamentous ascomycetes, and to allow conclusions about the evolution of fungal development, we sequenced the genome and assayed transcriptomes during development of Pyronema confluens, a saprobic Pezizomycete with a typical apothecium as fruiting body. With a size of 50 Mb and similar to 13,400 protein-coding genes, the genome is more characteristic of higher filamentous ascomycetes than the large, repeat-rich truffle genome; however, some typical features are different in the P. confluens lineage, e.g. the genomic environment of the mating type genes that is conserved in higher filamentous ascomycetes, but only partly conserved in P. confluens. On the other hand, P. confluens has a full complement of fungal photoreceptors, and expression studies indicate that light perception might be similar to distantly related ascomycetes and, thus, represent a basic feature of filamentous ascomycetes. Analysis of spliced RNA-seq sequence reads allowed the detection of natural antisense transcripts for 281 genes. The P. confluens genome contains an unusually high number of predicted orphan genes, many of which are upregulated during sexual development, consistent with the idea of rapid evolution of sex-associated genes. Comparative transcriptomics identified the transcription factor gene pro44 that is upregulated during development in P. confluens and the Sordariomycete Sordaria macrospora. The P. confluens pro44 gene (PCON_06721) was used to complement the S. macrospora pro44 deletion mutant, showing functional conservation of this developmental regulator

De novo Assembly of a 40 Mb Eukaryotic Genome from Short Sequence Reads: Sordaria macrospora, a Model Organism for Fungal Morphogenesis

Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30–90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in ∼4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for comparative studies to address basic questions of fungal biology