Genetic and Molecular Dissection of Hyphal Anastomosis

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Scientific merit. Filamentous fungi are the primary degraders of plant cell wall material in the environment. Some species are associated with the roots of plant species in a symbiotic relationship that allows plants to gain micronutrients from soil. Other fungal species are disease-causing agents in plants or animal/humans. Filamentous fungi, both beneficial and pathogenic species, grow by tip extension, branching and hyphal fusion to form an interconnected hyphal network. This hyphal network is believed to be essential for transfer of nutrients throughout ecosystems by root associated and saprophytic fungi and for colony establishment and exploitation of resources in pathogenic fungi. Studies on the filamentous fungus, Neurospora crassa, have revealed a complex and carefully regulated biological process of germling/hyphal fusion. During chemotropic interactions prior to cell fusion, published studies have shown that the alternating oscillation of signaling proteins from the tip of one fusion cell to its partner provides a compelling model for signaling between genetically identical cells. Physiological differences occur over a short time period and are associated with signal transduction feedback mechanisms. From these studies, it is evident that N. crassa uses a secreted chemical language to coordinate chemotropic interactions and cell fusion. The first objective of this project is to elucidate the signaling mechanisms and the language that filamentous fungi use to coordinate development of their hyphal network. Cell fusion is an important developmental process during mating and during tissue formation (i.e. the formation of multinucleate muscle cells and osteoclasts), although the mechanisms associated with cell-cell fusion are not well characterized. A second objective of this work is to identify additional components necessary for cell wall disassembly/membrane merger (fusogens). The ultimate goal of this work is to understand how the formation of the hyphal network allows a fungal individual to perform the physiological and developmental processes required to compete in nature and to complete its life cycle. Broader impact. Hyphal fusion in filamentous fungi is comparable to cell fusion events between genetically identical cells resulting in syncytia, such as myoblast fusion during muscle differentiation, trophoblast fusion during placental development and between osteoclasts during bone formation. Thus, studying cell-cell fusion in N. crassa provides a useful model for understanding molecular mechanisms of cell fusion events in more complex eukaryotic species. This project is an excellent training project as it incorporates live cell imaging techniques and microscopy, as well as genetics, biochemistry, genomics and molecular biology. It is expected that undergraduate researchers will be co-authors on research papers, as for previous NSF awards. The cell biological work on germling/hyphal fusion will continue collaborations with a mathematician interested in fluid dynamics, a bio-nanotechnologist, and amateur mycologists. A French television documentary that focuses on the analysis and function of a filamentous fungal hyphal network is currently being developed in collaboration with French documentarians.