Functional genomics of fungal morphogenesis: Projects

In the project group "functional genomics of fungal morphogenesis", we work on several aspects of molecular biology of fungal development. More detailed information can be found in several publications on these subjects. Students can participate in current research projects during "S-Modul" courses that are offered at the Department of Molecular and Cellular Botany. These are four- or six-week lab courses that can lead to bachelor- or master-projects.

Multicellular development like, for example, the development of fungal fruiting bodies, requires the coordinated expression of many genes. Gene expression must happen at the right time and in the right place (here, the right cells of the organism) to result in a functional fruiting body. Also, external signals like light, temperature, humidity, availability of nutrients and many others have to be taken into account and appropriate genetic programs have to be activated to cope with different conditions. During the last few years, methods like microarrays and RNA-seq have been developed that make it possible to monitor the expression of hundreds to thousands of genes at a time. We are currently working on projects to investigate differential gene expression in several filamentous fungi, as well as comparative genomics projects. Analyses like these can help us to unravel how a fungal genome controls multicellular development. In another line of research, we study the evolution of mating type loci in basidiomycetes. Mating type loci determine compatibility between strains as a prerequisite for sexual develoment.

1. Sequencing, assembly and annotation of the Sordaria macrospora genome

genome assembly size 39.8 Mb
chromosomes 7
number of scaffolds 584
protein-coding genes 9869
transcript from protein-coding genes 10014

2. Sequencing, assembly and annotation of the Pyronema confluens genome

genome assembly size 50 Mb
number of scaffolds 1588
scaffold N50 135 kb
protein coding genes 13369
average size of CDSs 1093 nt
average size of mRNAs 1483 nt
tRNA genes 605
% coding sequence 29.2
mtDNA size 191 kb

3. Functional genomics of fruiting body development in the filamentous fungus Sordaria macrospora

Sordaria life cycle

Sordaria macrospora is a filamentous ascomycete which is closely related to Neurospora crassa. But in contrast to Neurospora, Sordaria is homothallic, which means that a single strain produces fruiting bodies without the need for a partner of opposite mating type. The life cycle of S. macrospora is shown in the picture. It starts with an ascospore which germinates and produces a mycelium. Within a week, fruiting bodies develop in which asci are produced that contain eight ascospores each. The ascospores are ejected from the fruiting body and the cycle starts again.

localization of PRO44

This picture shows the localization of the PRO44 protein within developing fruiting bodies. PRO44 was fused to the green fluorescent protein (GFP) to visualize its localization by fluorescence microscopy, and the fusion construct was expressed under control of the pro44 promoter and terminator regions (left). As a control, a construct expressing only GFP (middle) and a construct expressing a fusion of a blue fluorescent protein with histone H2A (right, H2A localizes to the nucleus) were used. PRO44 localizes to the cell nucleus, but in developing fruiting bodies, it is preferentially present in the outer layers and not in the core of the fruiting body.

MA plots developmental mutants vs. wild type

As part of this study, we used a combination of laser microdissection (LM) and RNA-seq. LM enables us to isolate young fruiting bodies from the surrounding mycelium. We can then extract RNA from these protoperithecia and use this for RNA-seq. The picture shows MA plots, where the expression ratio for each gene in a certain comparison (e.g. mutant versus wild type) is plotted against the average expression for this gene for all analyzed genes. Genes shown in red are significantly up- or downregulated in the analyzed comparison.

phenotype of delta-asm2 mutant

One of the genes that were differentially regulated in mutant pro44 compared to the wild type, is asm2. This gene encodes a putative transcription factor, and its deletion results in defects in ascospore maturation, which can be seen in the figure to the left, where the wild type and the asm2 deletion mutant were grown for one to two weeks on different media (BMM and SWG). A complemented strain (third row for both time points) again shows the wild type phenotype.

4. Comparative functional genomics with Sordaria macrospora, Neurospora crassa, Pyronema confluens, and Ascodesmis nigricans

comparative expression analysis

This pictures shows the results of a first comparison of gene expression for nine genes (here identified by their numbers in each row) from P. confluens (P.c.) and S. macrospora (S.m.). For each gene, the ratio of gene expression during fruiting body development versus vegetative growth was determined in both fungi by quantitative real time PCR. The results are given as logarithmic values, i.e. positive values (red) indicate that a gene is upregulated during development and negative values (green) that it is downregulated. Values between -1 and +1 (gray) indicate that the gene is not differentially expressed. Among the nine genes, three are upregulated and another three are not differentially regulated in both species, indicating that there is a large degree of conservation of gene expression during fruiting body development even in these distantly related fungi.

On the left, you can see the results of a similar comparison, this time including all orthologous genes that were expressed in both organisms (several thousand genes) under several conditions. Due to the large number of genes, genes are not labelled individually any more. Also, this comparison does not show gene expression ratios like the picture above, but rather sequence read counts that were the results of RNA-seq experiments. This comparison was made as part of the Pyronema genome and transcriptome project described above. More details can be found in the genome publication.

Polyketide biosynthesis cluster

This picture shows one such gene cluster. The genomic organisation in S. macrospora (S.m.) and N. crassa (N.c.) is indicated, each gene is given as an arrow. The color indicates whether a gene is upregulated (red) or not upregulated (black or gray) during sexual development. Gene regions that are connected by gray shading between the two different species are similar, indicating that not only the expression patterns, but also the genomic organisation of this regions is largely conserved in these two different species. One of the genes (2925) was subsequently shown to be involved in fruiting body formation, because its deletion leads to slower maturation of fruiting bodies.

app expression

This picture shows that the APP protein accumulates only in fruiting bodies and not in the surrounding mycelium in both S. macrospora and N. crassa. The upper part shows the microscopic analysis of a fusion protein that consists of APP and the green fluorescent protein (GFP). The fusion construct was expressed under control of the app regulatory regions. The picture on the left shows a differential interference contrast (DIC) of a protoperithecium (young fruiting body) with surrounding hyphae, and the green fluorescence derived from GFP can be observed in the protoperithecium but not in the surrounding mycelium. Below, an analysis of the native APP protein from N. crassa is shown. In this case, protein extracts from fruiting bodies plus surrounding mycelium (total), from fruiting bodies alone (perithecia), and from the surrounding mycelium alone (mycelium) were extracted after 10, 12, and 14 days of growth and separated on a denaturing protein gel. The band that represents APP is indicated by an arrow. APP can be found in the total extract and in the fruiting bodies, but not in the surrounding mycelium. These and other analyses indicate that expression of app is conserved in S. macrospora and N. crassa in both time and space. One of our future goals is to elucidate to which degree gene expression is conserved at different levels during fungal development.

5. Analysis of the evolution of sexual development in the Tremellomycetes

model for mating type evolution

In the figure, you can see a model for the evolution of mating type loci in Tremellomycetes that was developed based on a comparison of the genomes of 24 Trichosporonales species. Genes at the MAT loci containing homeodomain transcription factor genes (HD locus) or pheromone and receptor genes (P/R locus) are shown in red/pink and blue, respectively. Genes involved in sexual development, but not originally part of a MAT locus are shown in green, other genes in grey. Only genes from the C. neoformans MAT locus that are also linked to the core MAT genes (STE3, HD genes) in the Trichosporon lineage are shown (STE11, STE12, STE20, IKS1, MYO2, RPL22), other genes present at the mating type loci are left out for clarity. In pathogenic Cryptococcus species and in the Trichosporonales, there was independent fusion of the mating type loci. Furthermore, there was independent loss of one HD gene per mating type locus. Additional details can be found in the publication in PLoS Genetics (Sun et al. 2019, PLoS Genet 15: e1008365).