A systems biology approach to dissect the function of the antifungal protein AnAFPfor Aspergillus niger

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The overall objective of the proposal is to gain mechanistic insights into the endogenous function of AnAFP for A. niger. Almost nothing is known about the biological function of Cys-stabilized antimicrobial peptides (AFPs) from filamentous fungi. Understanding the genetic, molecular and evolutionary basis of AFPs from fungi is important for understanding whether their function is mainly defensive or associated with fungal growth and development. In our previous work we uncovered that anafp expression parallels with the expression of autophagic proteins and processes and is selectively activated in highly vacuolated compartments of the vegetative mycelium, supposed to undergo autophagy for nutrient recycling. In view of the membrane-active potential of AnAFP and its subtle but lethal cell-wall stress provoking activity on A. niger, we propose that the tight spatial and temporal control of its gene expression enables AnAFP to fulfil an important intracellular function during nutrient starvation, i.e. during autophagic processes, thus contributing to the survival of A. niger during this life-threatening condition. Our working hypothesis is also encouraged by publications from others who demonstrated that asexual sporulation of A. nidulans is accompanied by autolytic and apoptotic processes. The key scientific aim of this proposal is to test this hypothesis. To achieve the Overall objective, we will exploit state-of-the-art Tools which have been established in our lab for A. niger including systems biology tools, synthetic biology tools, controlled bioreactor cultivations for physiological characterizations, confocal fluorescence microscopy to study AnAFP localisation and the fate of GFP-labelled organelles during autophagy as well as transmission electron microscopy to study the cellular ultrastructure of A. niger upon AFP exposure. Using such a holistic approach, which is embedded in four work packages (WP), the role of AnAFP for A. niger will be studied in detail to achieve the following four objectives: -Gain mechanistic insights into the timely and quantitatively controlled expression of AnAFP -Unravel the importance of AnAFP for cell survival and / or cell death -Unravel cellular targets of AnAFP and identify AnAFP interacting proteins-Refine the AnAFP co-expression gene network The ultimate goal is in understanding the mechanisms behind AnAFP-related processes that balance cell function and cell dysfunction, cell survival and cell death. Understanding the pathways and processes ensuring the tightrope walk between survival and death will on the one Hand ultimately identify an 'Achilles heel' of A. niger thus creating the possibility for targeted intervention of this or related pathogenic fungi from the genus Aspergillus. On the other hand, new leads for improved growth of A. niger during carbon starvation can be deduced which will increase the productivity of this cell factory during industrial fermentation.