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.

Neurospora 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.

Over 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.

We 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.

PP-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.

The 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.

Lastly, 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.

The overall goal of my scientific pursuits has been to understand how the environment influences ecological systems and organismal biology and vice versa. Over the past two centuries humans have placed unprecedented demands on the Earth’s natural resources. As a result of these pressures, we have witnessed an accelerated loss of biodiversity and a dramatic alteration of ecological patterns and processes. These changes have potentially devastating consequences in the long-term and are a cause for instigating novel scientific investigation and ecological mediation. Thus, the aim of my doctoral research was to understand community dynamics and primary metabolisms of soil-dwelling microbes following a fire disturbance.

Prescribed fire is a critical strategy for mitigating the effects of catastrophic wildfires. While the above-ground response to fire has been well-documented, fewer studies have addressed the effect of prescribed fire on soil microorganisms. As documented in Chapter 2, we set out to understand how soil microbial communities respond to prescribed fire. For this work we extensively sampled four plots (two burned, two controls) for 17 months in a mixed conifer forest in northern California, USA. Using amplicon sequencing, we found that prescribed fire significantly altered both fungal and bacterial community structure. We found that most differentially abundant fungal taxa had a positive fold-change, while differentially abundant bacterial taxa generally had a negative fold-change in burned plots. We tested the null hypothesis that these communities assembled due to neutral processes (i.e., drift and/or dispersal), finding that >90% of taxa fit this neutral prediction. However, a dynamic subcommunity composed of burn-associated indicator taxa that were positively differentially abundant was enriched for non-neutral ASVs, suggesting assembly via deterministic processes. In synthesizing these results, we identified 15 pyrophilous taxa with a significant and positive response to prescribed burns. Together, these results lay the foundation for building a process-driven understanding of microbial community assembly in the context of the classical disturbance regime of fire.

Fires have a significant impact on soil carbon stocks because combustion transforms carbon into either CO2 or in the production of recalcitrant pyrogenic organic matter (PyOM). PyOM is chemically heterogeneous and composed of a spectrum of fixed organic carbon (C), ranging from living plant biomass to completely charred material and soot. Pyrogenic carbon can modify microbial activity via promotion of microbial metabolism of PyOM as an energy or nutrient source, alteration of physical and chemical properties of the soil, or by interference of microbial signaling. As documented in Chapter 3, we were interested in bacterial species whose metabolic activities were promoted by the PyOM generated by fire. We targeted and isolated post-fire bacteria which can utilize the complex carbon compounds, found in PyOM, from fire-affected soils collected from wildfire sites across Northern California, in addition to soil collected from our prescribed burn sites. We find two bacterial genera, Streptomyces sp. and Paraburkholderia sp., were the dominate species in our post-fire isolate collection, suggesting they have the metabolic potential to utilize carbon substrates found in PyOM. Further, preliminary experiments reveal that a post-fire isolate from our prescribed burn sites, Paraburkholderia caledonica F3, can grow on aromatic carbon substrates in the lab. Finally, genomic analyses reveal that Paraburkholderia caledonica F3 possess the metabolic potential to degrade a variety of complex aromatic carbon compounds found in PyOM. These findings provide an exciting and promising outlook for the application of these post-fire bacteria for bioremediation efforts following fire.

Bioremediation utilizes the naturally occurring abilities of microorganisms (or plants) to break down harmful compounds and detoxify a polluted environment. Many of these organisms are native to these ecosystems, thus researchers are particularly interested in uncovering who these microorganisms are and how they can metabolically catabolize toxic compounds, such as aromatics and PAHs. While isolating organisms which are native to environments of interest poses its own challenges, we are still able to strategically and successful isolated these specialists in the laboratory. The arduous task arises in interpreting gene functions which have a phenotype under laboratory conditions. However, recent innovations in high-throughput analysis of mutant phenotypes have improved the rate at which researchers can predict gene functions. As documented in Chapter 4, we applied Random-Barcoded Transposon Sequencing to characterize the functional pathways of aromatic degradation in our laboratory isolate, Paraburkholderia caledonica F3. Here we validate the success of the generation of our Paraburkholderia caledonica F3 barcoded transposon insertion mutant library and propose fitness experiments with compounds of interest which are to follow.