Long-Distance Non-Self Recognition and Control of Intercellular Communication in Neurospora crassa

All multicellular organisms face a challenge: resources must be distributed throughout their bodies to ensure continued growth and coordinate cellular activities, but once resources are socialized, how can cells be coerced into working for the organism as a whole, rather than individually reproducing at the expense of the body they inhabit? Solutions to this freeloader problem usually rely on aligning the reproductive interests of all cells in a body by ensuring all are genetically identical. However, such solutions pose problems for organisms with indeterminate colonial growth habits, a lifestyle characterized by vegetative expansion, fragmentation, and reintegration of separated fragments. The filamentous fungus Neurospora crassa exemplifies this lifestyle. Its body is a cross-linked syncytial network produced by the fusion of many cells. Individual nuclei flow throughout the colony, making all the products of each nucleus potentially available to freeloaders. To keep the reproductive interests of all nuclei aligned, N. crassa must restrict fusion to genetically cells. The fungus prevents non-self fusion using an array of non-self recognition (NSR) systems./nNSR systems are encoded by polymorphic kind recognition loci. Each NSR locus enables cells to behave differently toward other individuals with the same haplotype than they do toward individuals with distinct haplotypes. At least two haplotypes at an NSR locus must exist in a population for the system to function. If many NSR loci are spread throughout an organisms genome, the net output of all NSR systems transitions to kin recognition; this allows an organism to identify genetically identical and distinct individuals with high fidelity. N. crassa encodes 15 confirmed NSR loci, with dozens more suspected based on sequencing and phenotypic investigations. Different NSR systems function at various stages in N. crassa’s life cycle, and during distinct phases of sociality. The first phase of sociality is called communication, and occurs when two cells detect each other and reorient their growth to intersect. The Determinant Of Communication (DOC) NSR system restricts communication between non-identical cells before they touch./nWe published our discovery of the DOC system in 2016. At the time, we knew haplotypes at the doc locus were necessary and sufficient to specify which cells will be recognized as self. We also reported that DOC-2 localized to the cellular periphery and DOC-1 oscillated between the cell bodies and growing tips of cells along with the MAK-2 complex during intercellular communication. We found the DOC system was not required for self-communication, implying it suppresses communication until a compatible communication group (CG) signal is received. Finally, we identified five distinct communication group haplotypes (CGHs) at the doc locus in an N. crassa population from Louisiana; doc alleles from different CGHs were less than 50% identical at the nucleotide level, and exhibited trans-species polymorphism among N. crassa and other members of its genus./nLittle else was certain regarding the DOC system. With neither characterized homologues nor identifiable functional domains, the DOC proteins remained particularly opaque. The primary goal of my doctoral work was to advance our understanding of the DOC system. I attempted to improve our model of DOC-mediated long-distance NSR by investigating how different doc genes and alleles interact in vivo, and evaluating the roles played by various regions of the DOC proteins in regulating communication. In pursuit of these aims, I spent considerable time and effort developing improved methods for quantifying intercellular communication./nAfter developing and validating a flow cytometry-based communication assay and analysis pipeline, I used this assay to evaluate the communication behavior of N. crassa strains expressing incompatible alleles of the doc genes, multiple incompatible DOC systems, truncated and otherwise manipulated alleles of the doc genes, and CGH1/CGH3 chimeric alleles of doc-1. The results of these experiments support the following model for DOC-mediated long-distance NSR:/nAs cells begin to communicate, general and CG-specific signals must be exchanged. CG signals are validated through interactions between the middle region of DOC-1 and DOC-2: matched CGH-variants of the DOC proteins are generally required for proper CG specificity. If the DOC system does not receive a compatible CG signal, DOC-1 and DOC-2 both prevent reinforcement of MAK-2 complex oscillations and suppress communication. This suppression involves the actions of the middle and C-terminal regions of DOC-1, and does not require the N-terminal region of DOC-2. If a compatible CG signal is detected, the DOC system allows MAK-2 complex oscillations and chemotropic growth to continue. DOC- 1’s N-terminal region is required to properly derepress communication, and is probably involved in validating CG signals.

Advisor: Glass, Nancy L