Filamentous fungi are the primary degraders of plant cellulosic material in the environment. Different species of filamentous fungi are associated with the roots of almost all plant species in a symbiotic relationship that allows plants to gain micronutrients from soil. In addition, other species are disease causing agents in plants, or cause important human diseases. All of these filamentous fungi, both beneficial and pathogenic species, grow by tip extension, branching and hyphal fusion to form a hyphal network. The formation of the 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. Although tip growth and branching have been extensively studied in filamentous fungi, little is known about the mechanism of hyphal fusion or the function of the hyphal network. Live cell imaging and genetic analysis of hyphal fusion in the model filamentous fungus, Neurospora crassa have revealed that the process of hyphal fusion is a complex and carefully regulated biological process. A large number of components essential for hyphal fusion have been identified in N. crassa, including components of signal transduction pathways, polarization components, filamentous fungal specific proteins and transmembrane proteins. Hyphal fusion mutants show developmental defects, in addition to slower growth rates and/or lag phase to reach maximal growth rate. These observations indicate that the formation of an interconnected network in filamentous fungi is essential for both exploitation of an environmental niche and for developmental processes. The objectives of this research are to dissect the process of germling/hyphal fusion using a combination of live cell imaging, genetic analysis and biochemical tools to elucidate the mechanisms of self-signaling, polarization and membrane merger with the ultimate goal of understanding the role of hyphal networks in ecosystems, symbiosis and pathogenesis.
Broader Impact: Hyphal fusion in filamentous fungi is comparable to cell fusion events between genetically identical cells in other organisms, such as cell-cell fusion 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 provides excellent training projects for undergraduate, graduate students and post-doctoral associates. A number of undergraduate California Opportunity Scholars have been recruited to this project; these students come from disadvantaged backgrounds and low performing schools in the Bay Area.
Scientific Merit: Filamentous fungi are the primary degraders of plant cellulosic 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 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 with obvious consequences to the fungal individual (http://glasslab.weebly.com/germling-and-hyphal-fusion.html). From these studies, it is evident that N. crassa uses a secreted chemical language to coordinate chemotropic interactions and cell fusion. A major goal of our work is to elucidate the signaling mechanisms and the language that filamentous fungi use to coordinate development of their hyphal network. During this grant award, we identified proteins involved in chemical signaling, and which encode a mitogen activated protein (MAP) kinase and a protein of unknown biochemical function (SO). The alternating oscillation of MAK-2 and SO from one germling tip to another during chemotropic interactions prior to cell fusion 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 (Fleissner et al., PNAS 2009). We have also identified additional components required for chemical signaling and MAK-2/SO oscillation to germling tips. Future work will elucidate the connection between these new signaling components and MAK-2 and SO. Following chemotropic interactions, fungal germlings undergo cell fusion via cell wall disassembly and plasma membrane merger. A second major goal of this work was to identify components necessary for cell wall disassembly/membrane merger (fusogens). We identified a transmembrane protein (PRM-1) that is involved in membrane merger in N. crassa and which localized to the site of fusion. Hyphal fusion in filamentous fungi is comparable to cell fusion events in animal cells that result in syncytia, for example, myoblast fusion during muscle differentiation, trophoblast fusion during placental development and between osteoclasts during bone formation. Proteins involved in membrane merger (fusogens) in these systems have not been elucidated. 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. Broader impacts: We have trained undergraduate, graduate students and post-doctoral associates on this project, which incorporates live cell imaging techniques and microscopy, as well as genetics, biochemistry, genomics and molecular biology. UCB undergraduate researchers are co-authors on research papers on germling/hyphal fusion funded by this NSF award. The cell biological work on germling/hyphal fusion is intellectually and visually compelling and has resulted in collaborations with a mathematician interested in fluid dynamics (Dr. Marcus Roper), a bio-nanotechnologist (Prof Dan Nicolau) and presentations to amateur mycologists in the Mycological Society of San Francisco and Bay Area Mycological Society.
