Fungi are rich sources of secondary metabolites (SMs) that are suggested to contribute to fundamental cellular processes like defense, communication with other (micro-) organisms, or virulence in pathogenic interactions. This is also true for Trichoderma mycoparasites, fungi that are able to attack and parasitize other fungi. However, most SM-related gene clusters are not expressed under standard cultivation conditions but only when exogenous chemical cues are present. This project aims at elucidating the role of chemical cross-talk by small molecules in triggering metabolite production and in mediating the interaction between two fungi, the mycoparasite Trichoderma atroviride and the plant pathogen Botrytis cinerea. We will develop and employ a novel integrative approach for in situ detection and for the first time spatial localization of small molecules produced and exchanged between two filamentous fungi. The biological role of small molecule triggers on SM production in the respective interaction partners will be studied at various cellular levels by applying a combination of cutting-edge technologies that is unparalleled in the field. The tailormade integration of mass spectrometric imaging, isotope-assisted metabolomics, live cell imaging, and RNA-sequencing will provide novel insight into complex mycoparasitic interactions between the tested filamentous fungi and the identity and role of small molecules therein.