With a combination of microscopic laser scissors and modern sequencing methods, biologists at the Ruhr-Universität have analyzed the activity of genes in the entire genome of certain fungi in one fell swoop. Especially with organisms in the millimetre size range, it is a particular challenge because little cell material is available. The scientists of the RUB Department of General and Molecular Botany took advantage of the method to investigate the development of small multicellular fungi.
The results are reported in the journal BMC Genomics.
Gene activity differs from tissue to tissue
In multicellular organisms, each cell contains the same genetic material, however, often only a fraction of the genes are active (expressed). These differences in gene expression are the cause of variations in the structure and physiology of cells. Gene expression is therefore the key to understanding the development of multicellular organisms. "In large organisms such as plants, it is usually not a problem to get enough starting material to study gene expression," explains Dr. Minou Nowrousian. "In the case of microorganisms, organs often consist of only a few cells, and might be embedded in other tissues from which they are difficult to separate." Therefore, biologists of the research groups of Prof. Dr. Ulrich Kück and Minou Nowrousian combined laser microdissection with modern sequencing technologies to analyze the gene activity during the development of certain just 0.5 millimetres large sexual structures of fungi.
How laser microdissection works
In laser microdissection, scientists cut defined regions of a sample under the light microscope with a laser beam. With this laser mini-scissors, the RUB researchers collected the fruiting bodies, i.e. the sexual structures of the fungus Sordaria macrospora, which has been used for decades as a model organism in developmental biology. From the fruiting bodies, they isolated the RNA which represents the gene activity. With the help of "next generation" sequencing, they characterized the activity of all genes of the genome simultaneously.
A transcription factor controls genetic activity in young fruiting bodies
The Bochum researchers compared the wild-type fungus with a mutant form that has no mature fruiting bodies, in other words is not able to reproduce sexually. For this purpose, they studied gene expression in young, immature fruiting bodies. They showed that some fruiting body-specific genes are not activated in the mutant. The defective gene contains the "building instructions" for a so-called transcription factor - a protein that turns other genes on or off. The RUB team also found that the fruiting body has a completely different genetic activity pattern to non-reproductive tissue. "With the new combination of methods, we want to investigate the activity of genes in other mutants and developmental stages to better understand the molecular mechanisms of multicellular development in fungi," said Prof. Kück.
Fungi: ecological and economic importance
Fungi have a big impact on virtually all ecosystems. They make significant contributions to the reduction of animal and vegetable waste products and thereby contribute to the global carbon cycle. Some species live in symbiosis with plants or animals, other species are pathogens. In the chemical and pharmaceutical industry, fungi are used for the production of antibiotics and enzymes. The formation of pathogenic or symbiotic interactions and the production of medicines or biotechnology-related substances are often tied to specific stages in the life cycle of a fungus. The analysis of the development is therefore crucial not only for basic research but also for industrial applications.
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I. Teichert, G. Wolff, U. Kück, M. Nowrousian (2012): Combining laser microdissection and RNA-seq to chart the transcriptional landscape of fungal development, BMC Genomics, doi: 10.1186/1471-2164-13-511