Doktoratskolleg MEIBio - Molecular and Elemental Imaging in Bioscience
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bernhard.seiboth(a)tuwien.ac.at

PhD Fellow #3

Quantitative mapping of localized cellulase expression and secretion in the functionally stratified fungal colony by combined mass spectrometry and live-cell imaging

Faculty: Bernhard Seiboth

Many fungi are saprobic chemoheterotrophic organisms which degrade the organic matter in their surrounding by extracellular enzymatic digestion. Apical growth is a hallmark of filamentous fungi and accounts for much of the success of this group of organisms. This tip growth is a specialized form of polar growth and results in elongated hyphal cells which explore the environment for nutrients. On a substrate with homogenous nutrient distribution fungi form a radial colony which differentiates distinct zones with 'tissue-like', specific developmental and metabolic functions. The genetic and molecular basis of this functional stratification, however, is still unknown. Which regions of the mycelial colony recognize the available carbon source, in which regions are the degrading enzymes synthetized and where are they secreted, or which areas of the colony are the preferred sites of the uptake of degradation products for intracellular energy production?

The basic questions are addressed within the proposed project by multimodal imaging employing mass spectrometry (MS) to monitor changes of carbon content and protein composition in the growing colony and the substrate in order to correlate key processes of substrate degradation with developmental specialization of the colony. As a model system we will use the ascomycete Trichoderma reesei and cellulose, the most abundant polymer on earth, as substrate.

Proteins involved in cellulose metabolism will be localized in vivo in the fungal colony (Figure) including the cellulases themselves and key factors involved in cellulase regulation, such as transcription, translation, and secretion, and or the uptake of the cellulose degradation products. MS will furthermore lead to the identification of yet unknown key proteins of fungal cellulose catabolism. Additionally, novel regulators and systemic signalling proteins will be identified through pull-down binding assays using known regulators as bait proteins. The newly identified key proteins will be functionally characterized and localized by live-cell imaging in the fungal colony. Dynamics of fluorescently labelled target proteins will be monitored throughout the colony network and functional localization patterns on varying carbon sources assessed.

Collaborations:

  • Collaborations with the CBS-KNAW Fungal Biodiversity Centre (Dr. Ronald de Vries) are documented.
  • Multimodal Imaging will be approached by combining fluorescence spectroscopy and mass spectrometry together with G. Allmaier and M. Marchetti-Deschmann (TU Vienna).
  • Attempts to align data from live-cell imaging experiments with mass spectrometric data will be addressed by R. Sablatnig (TU Vienna).
  • High-end live-cell imaging will be conducted in collaboration with microscopy facilities at the University of St. Andrews, Scotland (Dr. Jens Tilsner).