Mechanisms of communication between genetically identical cells in Neurospora crassa

Cell-to-cell communication and cell fusion are fundamental biological processes across the tree of life. Abundant research has unraveled the mechanisms that mediate cell-to-cell communication in diverse organisms from bacteria to humans. However much of this research has focused on cell-to-cell communication between fundamentally different cells (i.e. host-pathogen interactions), or population level density-related communication among similar cells (i.e. quorum sensing). This dissertation describes a different form of cell-to-cell communication that occurs between genetically identical cells./nNeurospora crassa is a filamentous Ascomycete fungus, and a well-developed model organism for studying eukaryotic genetics and cell biology, including cell-to-cell communication that results in cell fusion. N. crassa is a particularly compelling model for studying communication-mediated cell fusion because this process is important during three different stages in the life cycle. First, shortly after the asexual spores germinate into germlings, they will chemotropically grow toward and ultimately fuse with other genetically identical individuals. Germling fusion is an important aspect of colony establishment. Second, after a colony is established, communication and cell fusion will continue to occur between hyphae within a single colony. Hyphal fusion is important for maintaining and reinforcing a robust colony. Germling and hyphal fusion represent two different types of asexual cell fusion, in which communication occurs between genetically identical individuals resulting in cell fusion and a shared cytoplasm. There is no nuclear fusion or genetic recombination associated with asexual cell fusion. The third form of communication and cell fusion in N. crassa is classic sexual cell fusion that occurs between the female trichogyne and a male cell of opposite mating type. After sexual cell fusion, nuclear fusion and genetic recombination occurs. Sexual cell fusion also triggers the development of the fruiting body, which is called a perithecium. This dissertation focuses on unraveling the mechanism by which two genetically identical cells communicate and fuse (i.e. germling or hyphal fusion), and differentiating this process from sexual communication and cell fusion./nOver seventy genes have been identified as involved in the process of germling communication and cell fusion. However, the vast majority of these genes do not make up any known regulatory network. Two evolutionarily conserved Mitogen Activated Protein Kinase (MAPK) pathways are necessary for germling communication; the MAK-1/Cell Wall Integrity (CWI) pathway, and the MAK-2 signal response pathway. The MAK-2 pathway is orthologous to the pheromone response pathway in Saccharomyces cerevisiae, which like most MAPK pathways, begins with signal reception and ends in a transcriptional response. By expressing a luciferase reporter in each communication mutant, we were able to determine which genes broadly function upstream of the MAK-2 pathway, and which genes broadly function downstream. In general, these data correlated with the inherent fusion frequency of each mutant. The PP-1 transcription factor protein is the conserved downstream target of the MAK-2 MAPK pathway, and PP-1 directly regulates transcription of our luciferase reporter. We confirmed that a second transcription factor, ADV-1, is also required for expression of our luciferase reporter. Lastly, we identified an unexpected small group of mutants with moderate luciferase expression (including Δham-11), which indicated that these knocked out genes are either involved in a positive feedback loop that reinforces signaling, or more than one pathway exists upstream of the MAK-2 pathway./nWe leveraged the unique phenotype of the Δham-11 mutant to explore the broad nature of germling communication and identify candidate signaling-mutants. Δham-11 germlings do not normally communicate on their own, but Δham-11 germlings will communicate and fuse with wild-type germlings. By co-culturing the Δham-11 mutant with other communication mutants, we identified several mutants that Δham-11 germlings can communicate with, and several mutants that that do not communicate with Δham-11 germlings. To our surprise, we observed Δham-11 germlings chemotropically growing toward Δsoft germlings, but Δsoft germlings never responded and fusion never occurred. The SOFT protein is largely uncharacterized, but is hypothesized to facilitate signal sending. In contrast to this hypothesis, our data indicate that Δsoft germlings are capable of producing a signal that elicits a chemotropic response from Δham-11 germlings./nPP-1 is the evolutionarily conserved target of the MAK-2 pathway, but it was unclear how ADV-1 and the CWI pathway are also involved in regulating germling communication and cell fusion. To identify transcriptional targets, we did RNAseq on Δpp-1, Δadv-1, and wild-type germlings in addition to DNA Affinity Purification sequencing (DAP-seq) on PP-1 and ADV-1 proteins in vitro. These data indicated that PP-1 regulates transcription of adv-1, and then ADV-1 directly regulates transcription of many of the genes required for communication, adhesion, fusion, non-self recognition, growth, development, and stress response. Furthermore, misexpression of adv-1 completely suppressed the pleiotropic phenotype of the Δpp-1 mutant, but the inverse was not true. In an effort to elucidate the regulatory network, we misexpressed adv-1 and pp-1 in the putative upstream Δmak-1 and Δmak-2 mutants. Misexpressed pp-1 did not rescue the phenotype of either MAPK mutant, and misexpression of adv-1 only partially rescued the growth phenotype of Δmak-1 cells. Together our data indicate that MAK-1 is required for full activation of MAK-2, MAK-2 is required for activation or de-repression of PP-1, PP-1 directly regulates transcription of adv-1, and then ADV-1 directly regulates transcription of target genes. Additionally, our data indicate a link between MAK-1 and ADV-1 that is independent of PP-1./nThe protein HAM-5 was recently identified at the scaffold for the MAK-2 pathway. We further characterized functional domains of the HAM-5 protein and identified a protein, HAM-14, that may be important for mediating the formation of a HAM-5/MAK-2-pathway protein complex. HAM-14 physically interacts with members of the MAK-2 cascade via co-immunoprecipitation, but HAM-14 does not show the same dynamic fluorescent localization as the HAM-5/MAK-2 complex. The Δham-14 mutant is completely defective at germling communication and fusion, but not hyphal fusion or sexual development, which is reminiscent of the phenotype of a Δham-11 mutant./nLastly, we demonstrate an interesting genetic interaction between the ham-11 gene and the doc-1 and doc-2 genes. The doc-1 and doc-2 genes define “dialects” or communication groups (CG) among wild strains of N. crassa. The Δdoc-1 Δdoc-2 mutant grown on its own has a phenotype that is equivalent to wild-type, however Δdoc-1 Δdoc-2 germlings will not communicate with wild-type germlings, even though they are completely isogenic except for lacking the doc-1 and doc-2 genes. Surprisingly, Δham-11 germlings are able to communicate and fuse with Δdoc-1 Δdoc-2 germlings. To investigate the interaction between these genes we made a Δdoc-1 Δdoc-2; Δham-11 mutant. This triple mutant has a remarkably synthetic and specific phenotype that is completely defective at all forms of asexual communication and fusion, but growth and sexual reproduction are unaffected. Together our data indicate that ham-11 and doc-1-doc-2 function in parallel and upstream of the MAK-2 pathway to mediate asexual communication by integrating self and non-self recognition systems.

Advisor: Glass, N. Louise