A rapid PCR-based method to determine the Neurospora crassa mating type.

So far mating type determination in Neurospora crassa requires test crosses with strains of known mating type. We present a simple, quick, and reliable polymerase chain reaction-based method for mating type determination in N. crassa.

Insights into Taxol® biosynthesis by endophytic fungi.

There have been two hundred reports that endophytic fungi produce Taxol®, but its production yield is often rather low. Although considerable efforts have been made to increase Taxol/taxanes production in fungi by manipulating cocultures, mutagenesis, genome shuffles, and gene overexpression, little is known about the molecular signatures of Taxol biosynthesis and its regulation. It is known that some fungi have orthologs of the Taxol biosynthetic pathway, but the overall architecture of this pathway is unknown. A biosynthetic putative gene homology approach, combined with genomics and transcriptomics analysis, revealed that a few genes for metabolite residues may be located on dispensable chromosomes. This review explores a number of crucial topics (i) finding biosynthetic pathway genes using precursors, elicitors, and inhibitors; (ii) orthologs of the Taxol biosynthetic pathway for rate-limiting genes/enzymes; and (iii) genomics and transcriptomics can be used to accurately predict biosynthetic putative genes and regulators. This provides promising targets for future genetic engineering approaches to produce fungal Taxol and precursors. KEY POINTS: • A recent trend in predicting Taxol biosynthetic pathway from endophytic fungi. • Understanding the Taxol biosynthetic pathway and related enzymes in fungi. • The genetic evidence and formation of taxane from endophytic fungi.

Complementation of an Eisosomal Yeast pil1 Mutant and Characteristics of Eisosomal Distribution in Hyphae of Neurospora crassa Germinating from Two Different Spore Types.

Eisosomes are plasma-membrane-associated protein complexes of fungi and algae involved in various cellular processes. The eisosome composition of the budding yeast is well described, but there is a limited number of studies only about eisosomes in filamentous fungi. In our study, we examined the Neurospora crassa LSP-1 protein (NcLSP1). By complementing a Saccharomyces cerevisiae Δpil1 mutant strain with nclsp1, we show the functional homology of the NcLSP1 to yeast PIL1 rather than to yeast LSP1 and hereby confirm that the NcLSP1 is an eisosomal core protein and suitable eisosomal marker. The subsequent cloning and expression of the nclsp1::trfp reporter gene construct in N. crassa allowed for a systematical investigation of the characteristics of eisosome formation and distribution in different developmental stages. In N. crassa, the hyphae germinating from sexual and asexual spores are morphologically identical and have been historically recognized as the same type of cells. Here, we demonstrate the structural differences on the cellular level between the hyphae germinating from sexual and asexual spores.

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 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.

Transcriptomic analysis of poco1, a mitochondrial pentatricopeptide repeat protein mutant in Arabidopsis thaliana.

BACKGROUND: Flowering is a crucial stage during plant development. Plants may respond to unfavorable conditions by accelerating reproductive processes like flowering. In a recent study, we showed that PRECOCIOUS1 (POCO1) is a mitochondrial pentatricopeptide repeat (PPR) protein involved in flowering time and abscisic acid (ABA) signaling in Arabidopsis thaliana. Here, we use RNA-seq data to investigate global gene expression alteration in the poco1 mutant. RESULTS: RNA-seq analysis was performed during different developmental stages for wild-type and poco1 plants. The most profound differences in gene expression were found when wild-type and poco1 plants of the same developmental stage were compared. Coverage analysis confirmed the T-DNA insertion in POCO1, which was concomitant with truncated transcripts. Many biological processes were found to be enriched. Several flowering-related genes such as FLOWERING LOCUS T (FT), which may be involved in the early-flowering phenotype of poco1, were differentially regulated. Numerous ABA-associated genes, including the core components of ABA signaling such as ABA receptors, protein phosphatases, protein kinases, and ABA-responsive element (ABRE) binding proteins (AREBs)/ABRE-binding factors (ABFs) as well as important genes for stomatal function, were mostly down-regulated in poco1. Drought and oxidative stress-related genes, including ABA-induced stress genes, were differentially regulated. RNA-seq analysis also uncovered differentially regulated genes encoding various classes of transcription factors and genes involved in cellular signaling. Furthermore, the expression of stress-associated nuclear genes encoding mitochondrial proteins (NGEMPs) was found to be altered in poco1. Redox-related genes were affected, suggesting that the redox state in poco1 might be altered. CONCLUSION: The identification of various enriched biological processes indicates that complex regulatory mechanisms underlie poco1 development. Differentially regulated genes associated with flowering may contribute to the early-flowering phenotype of poco1. Our data suggest the involvement of POCO1 in the early ABA signaling process. The down-regulation of many ABA-related genes suggests an association of poco1 mutation with the ABA signaling deficiency. This condition further affects the expression of many stress-related, especially drought-associated genes in poco1, consistent with the drought sensitivity of poco1. poco1 mutation also affects the expression of genes associated with the cellular regulation, redox, and mitochondrial perturbation.

Genomics- and Metabolomics-Based Investigation of the Deep-Sea Sediment-Derived Yeast, Rhodotorula mucilaginosa 50-3-19/20B.

Red yeasts of the genus Rhodotorula are of great interest to the biotechnological industry due to their ability to produce valuable natural products, such as lipids and carotenoids with potential applications as surfactants, food additives, and pharmaceuticals. Herein, we explored the biosynthetic potential of R. mucilaginosa 50-3-19/20B collected from the Mid-Atlantic Ridge using modern genomics and untargeted metabolomics tools. R. mucilaginosa 50-3-19/20B exhibited anticancer activity when grown on PDA medium, while antimicrobial activity was observed when cultured on WSP-30 medium. Applying the bioactive molecular networking approach, the anticancer activity was linked to glycolipids, namely polyol esters of fatty acid (PEFA) derivatives. We purified four PEFAs (1-4) and the known methyl-2-hydroxy-3-(1H-indol-2-yl)propanoate (5). Their structures were deduced from NMR and HR-MS/MS spectra, but 1-5 showed no anticancer activity in their pure form. Illumina-based genome sequencing, de novo assembly and standard biosynthetic gene cluster (BGC) analyses were used to illustrate key components of the PEFA biosynthetic pathway. The fatty acid producing BGC3 was identified to be capable of producing precursors of PEFAs. Some Rhodotorula strains are able to convert inulin into high-yielding PEFA and cell lipid using a native exo-inulinase enzyme. The genomic locus for an exo-inulinase enzyme (g1629.t1), which plays an instrumental role in the PEFA production via the mannitol biosynthesis pathway was identified. This is the first untargeted metabolomics study on R. mucilaginosa providing new genomic insights into PEFA biosynthesis.

PRECOCIOUS1 (POCO1), a mitochondrial pentatricopeptide repeat protein affects flowering time in Arabidopsis thaliana.

Flowering is a vital developmental shift in plants from vegetative to reproductive phase. The timing of this shift is regulated by various linked genetic pathways including environmental cues and internal regulation. Here we report a role for an Arabidopsis gene, AT1G15480, which encodes a P-class pentatricopeptide repeat (PPR) protein, affecting flowering time. We show that AT1G15480 is localized to mitochondria. An AT1G15480 T-DNA insertion line exhibits an early-flowering phenotype, which is quite a rare phenotype among PPR mutants. The early-flowering phenotype was observed under both long and short days compared with wild type plants. Genetic complementation confirmed the observed phenotype. We therefore named the PPR protein PRECOCIOUS1 (POCO1). poco1 plants showed lower respiration, ATP content and higher accumulation of superoxide. Importantly, the quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that the expression of FLOWERING LOCUS C (FLC), which is a key floral repressor, was strongly downregulated in the poco1. Likewise, the expression level of the FLC positive regulator ABSCISIC ACID-INSENSITIVE 5 (ABI5) was reduced in the poco1. Consistent with the qRT-PCR results, poco1 plants showed reduced sensitivity to abscisic acid compared with wild type with respect to primary root growth and days to flowering. Furthermore, the poco1 mutation enhances the sensitivity to drought stress. Further analysis showed that POCO1 affects mitochondrial RNA editing. Taken together, our data demonstrate a remarkable function of POCO1 in flowering time and the abscisic acid signalling pathway.

Genome Sequencing and analyses of Two Marine Fungi from the North Sea Unraveled a Plethora of Novel Biosynthetic Gene Clusters.

Marine Fungi are potent secondary metabolite producers. However, limited genetic information are available their biosynthetic gene clusters (BGCs) and their biotechnological applications. To overcome this lack of information, herein, we used next-generation sequencing methods for genome sequencing of two marine fungi, isolated from the German Wadden Sea, namely Calcarisporium sp. KF525 and Pestalotiopsis sp. KF079. The assembled genome size of the marine isolate Calcarisporium sp. KF525 is about 36.8 Mb with 60 BGCs, while Pestalotiopsis sp. KF079 has a genome size of 47.5 Mb harboring 67 BGCs. Of all BGCs, 98% and 97% are novel clusters of Calcarisporium sp. and Pestalotiopsis sp., respectively. Only few of the BGCs were found to be expressed under laboratory conditions by RNA-seq analysis. The vast majority of all BGCs were found to be novel and unique for these two marine fungi. Along with a description of the identified gene clusters, we furthermore present important genomic features and life-style properties of these two fungi. The two novel fungal genomes provide a plethora of new BGCs, which may have biotechnological applications in the future, for example as novel drugs. The genomic characterizations will provide assistance in future genetics and genomic analyses of marine fungi.

Characterization of indole-3-pyruvic acid pathway-mediated biosynthesis of auxin in Neurospora crassa.

Plants, bacteria and some fungi are known to produce indole-3-acetic acid (IAA) by employing various pathways. Among these pathways, the indole-3-pyruvic acid (IPA) pathway is the best studied in green plants and plant-associated beneficial microbes. While IAA production circuitry in plants has been studied for decades, little is known regarding the IAA biosynthesis pathway in fungal species. Here, we present the first data for IAA-producing genes and the associated biosynthesis pathway in a non-pathogenic fungus, Neurospora crassa. For this purpose, we used a computational approach to determine the genes and outlined the IAA production circuitry in N. crassa. We then validated these data with experimental evidence. Here, we describe the homologous genes that are present in the IPA pathway of IAA production in N. crassa. High-performance liquid chromatography and thin-layer chromatography unambiguously identified IAA, indole-3-lactic acid (ILA) and tryptophol (TOL) from cultures supplemented with tryptophan. Deletion of the gene (cfp) that encodes the enzyme indole-3-pyruvate decarboxylase, which converts IPA to indole-3-acetaldehyde (IAAld), results in an accumulation of higher levels of ILA in the N. crassa culture medium. A double knock-out strain (Δcbs-3;Δahd-2) for the enzyme IAAld dehydrogenase, which converts IAAld to IAA, shows a many fold decrease in IAA production compared with the wild type strain. The Δcbs-3;Δahd-2 strain also displays slower conidiation and produces many fewer conidiospores than the wild type strain.

Fungal genotype determines survival of Drosophila melanogaster when competing with Aspergillus nidulans.

Fungi produce an astonishing variety of secondary metabolites, some of which belong to the most toxic compounds in the living world. Several fungal metabolites have anti-insecticidal properties which may yield advantages to the fungus in competition with insects for exploitation of environmental resources. Using the Drosophila melanogaster/Aspergillus nidulans ecological model system to assess secondary metabolite mutant genotypes, we find a major role for the veA allele in insect/fungal confrontations that exceeds the influence of other factors such as LaeA. VeA along with LaeA is a member of a transcriptional complex governing secondary metabolism in A. nidulans. However, historically a mutant veA allele, veA1 reduced in secondary metabolite output, has been used in many studies of this model organism. To test the significance of this allele in our system, Aspergillus nidulans veA wild type, veA1, ΔveA and ΔlaeA were evaluated in confrontation assays to analyze egg laying activity, and the survival rate of larvae. The veA1 genetic background led to a significant increase of larval survival. Adult flies were observed almost exclusively on veA1, ΔveA or ΔlaeA genetic backgrounds, suggesting a role for the velvet complex in insect/fungal interactions. This effect was most profound using the veA1 mutant. Hence, larval survival in confrontations is highly affected by the fungal genotype.

An inducible tool for random mutagenesis in Aspergillus niger based on the transposon Vader.

The ascomycete Aspergillus niger is widely used in the biotechnology, for instance in producing most of the world's citric acid. It is also known as a major food and feed contaminant. While generation of gene knockouts for functional genomics has become feasible in ku70 mutants, analyzing gene functions or metabolic pathways remains a laborious task. An unbiased transposon-based mutagenesis approach may aid this process of analyzing gene functions by providing mutant libraries in a short time. The Vader transposon is a non-autonomous DNA-transposon, which is activated by the homologous tan1-transposase. However, in the most commonly used lab strain of A. niger (N400 strain and derivatives), we found that the transposase, encoded by the tan1 gene, is mutated and inactive. To establish a Vader transposon-based mutagenesis system in the N400 background, we expressed the functional transposase of A. niger strain CBS 513.88 under the control of an inducible promoter based on the Tet-on system, which is activated in the presence of the antibiotic doxycycline (DOX). Increasing amounts of doxycycline lead to higher Vader excision frequencies, whereas little to none activity of Vader was observed without addition of doxycycline. Hence, this system appears to be suitable for producing stable mutants in the A. niger N400 background.

De Novo Assembly and Genome Analyses of the Marine-Derived Scopulariopsis brevicaulis Strain LF580 Unravels Life-Style Traits and Anticancerous Scopularide Biosynthetic Gene Cluster.

The marine-derived Scopulariopsis brevicaulis strain LF580 produces scopularides A and B, which have anticancerous properties. We carried out genome sequencing using three next-generation DNA sequencing methods. De novo hybrid assembly yielded 621 scaffolds with a total size of 32.2 Mb and 16298 putative gene models. We identified a large non-ribosomal peptide synthetase gene (nrps1) and supporting pks2 gene in the same biosynthetic gene cluster. This cluster and the genes within the cluster are functionally active as confirmed by RNA-Seq. Characterization of carbohydrate-active enzymes and major facilitator superfamily (MFS)-type transporters lead to postulate S. brevicaulis originated from a soil fungus, which came into contact with the marine sponge Tethya aurantium. This marine sponge seems to provide shelter to this fungus and micro-environment suitable for its survival in the ocean. This study also builds the platform for further investigations of the role of life-style and secondary metabolites from S. brevicaulis.

Identification of the Scopularide Biosynthetic Gene Cluster in Scopulariopsis brevicaulis.

Scopularide A is a promising potent anticancer lipopeptide isolated from a marine derived Scopulariopsis brevicaulis strain. The compound consists of a reduced carbon chain (3-hydroxy-methyldecanoyl) attached to five amino acids (glycine, l-valine, d-leucine, l-alanine, and l-phenylalanine). Using the newly sequenced S. brevicaulis genome we were able to identify the putative biosynthetic gene cluster using genetic information from the structurally related emericellamide A from Aspergillus nidulans and W493-B from Fusarium pseudograminearum. The scopularide A gene cluster includes a nonribosomal peptide synthetase (NRPS1), a polyketide synthase (PKS2), a CoA ligase, an acyltransferase, and a transcription factor. Homologous recombination was low in S. brevicaulis so the local transcription factor was integrated randomly under a constitutive promoter, which led to a three to four-fold increase in scopularide A production. This indirectly verifies the identity of the proposed biosynthetic gene cluster.

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.

Deletions in cox2 mRNA result in loss of splicing and RNA editing and gain of novel RNA editing sites.

As previously demonstrated, the maize cox2 RNA is fully edited in cauliflower mitochondria. Use of constructs with a deleted cox2 intron, however, led to a loss of RNA editing at almost all editing sites, with only a few sites still partially edited. Likewise, one deletion in exon 1 and three in exon 2 abolish RNA editing at all cox2 sites analyzed. Furthermore, intron splicing is abolished using these deletions. Mutation of a cytosine residue, which is normally edited and localized directly adjacent to the intron, to thymidine did not result in restoration of splicing, indicating that the loss of splicing was not due to loss of RNA editing. One deletion in exon 2 did not lead to loss of splicing. Instead, most editing sites were found to be edited, only three were not edited. Unexpectedly, we observed additional RNA editing events at new sites. Thus it appears that deletions in the cox2 RNA sequence can have a strong effect on RNA processing, leading to loss of splicing, loss of editing at all sites, or even to a gain of new editing sites. As these effects are not limited to the vicinity of the respective deletions, but appear to be widespread or even affect all editing sites, they may not be explained by the loss of PPR binding sites. Instead, it appears that several parts of the cox2 transcript are required for proper RNA processing. This indicates the roles of the RNA sequence and structural elements in the recognition of the editing sites.

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 ~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.

Characterization of bud emergence 46 (BEM46) protein: sequence, structural, phylogenetic and subcellular localization analyses.

The bud emergence 46 (BEM46) protein from Neurospora crassa belongs to the α/ß-hydrolase superfamily. Recently, we have reported that the BEM46 protein is localized in the perinuclear ER and also forms spots close by the plasma membrane. The protein appears to be required for cell type-specific polarity formation in N. crassa. Furthermore, initial studies suggested that the BEM46 amino acid sequence is conserved in eukaryotes and is considered to be one of the widespread conserved "known unknown" eukaryotic genes. This warrants for a comprehensive phylogenetic analysis of this superfamily to unravel origin and molecular evolution of these genes in different eukaryotes. Herein, we observe that all eukaryotes have at least a single copy of a bem46 ortholog. Upon scanning of these proteins in various genomes, we find that there are expansions leading into several paralogs in vertebrates. Usingcomparative genomic analyses, we identified insertion/deletions (indels) in the conserved domain of BEM46 protein, which allow to differentiate fungal classes such as ascomycetes from basidiomycetes. We also find that exonic indels are able to differentiate BEM46 homologs of different eukaryotic lineage. Furthermore, we unravel that BEM46 protein from N. crassa possess a novel endoplasmic-retention signal (PEKK) using GFP-fusion tagging experiments. We propose that three residues namely a serine 188S, a histidine 292H and an aspartic acid 262D are most critical residues, forming a catalytic triad in BEM46 protein from N. crassa. We carried out a comprehensive study on bem46 genes from a molecular evolution perspective with combination of functional analyses. The evolutionary history of BEM46 proteins is characterized by exonic indels in lineage specific manner.

Alternative splicing in ascomycetes.

Alternative splicing is a complex and regulated process, which results in mRNA with different coding capacities from a single gene. Extend and types of alternative splicing vary greatly among eukaryotes. In this review, I focus on alternative splicing in ascomycetes, which in general have significant lower extend of alternative splicing than mammals. Yeast-like species have low numbers of introns and consequently alternative splicing is lower compared to filamentous fungi. Several examples from single studies as well as from genomic scale analysis are presented, including a survey of alternative splicing in Neurospora crassa. Another focus is regulation by riboswitch RNA and alternative splicing in a heterologous system, along with putative protein factors involved in regulation.