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In eukaryotes, different cell morphologies are generated by fine-tuning the spatiotemporal regulation of the polarized growth machinery. Studying hyphal growth in the multimorphic opportunistic yeast pathogen Candida albicans provides a unique opportunity to understand how highly polarized cell structures are generated and maintained, and has the potential to provide insight into mechanisms of pathogenesis. Hyphal cell morphology requires that polarized growth machinery be held at hyphal tips over extreme distances and through multiple cell cycles. Deletion of the bud-site selection GTPase Rsr1 in C. albicans results in defects in cell size and shape not observed in studies of its ortholog in the related yeast Saccharomyces cerevisiae. This suggests that, in addition to its role in bud site selection, Rsr1 has expanded function in C. albicans, which impacts polarized growth and the generation of the hyphal morphology. Here, I show that loss of Rsr1 results in changes to a hyphal-specific tip structure, the Spitzenkörper, a downstream developmental indicator of Cdc42 signaling, and key regulator of polarized growth. Also, my results show that Rsr1's function impacts the spatiotemporal distribution of Bem1 a marker of the active form of Cdc42. Interestingly, the changes in the distribution of Cdc42 activity are also correlated with reduced expression of the hyphal transcriptional program. In addition, I also show the differential effects of the guanine nucleotide binding states of Rsr1, through the manipulation of the Rsr1 GAP, Bud2, and GEF, Bud5. Through the action of Bud2, Rsr1-GDP acts as a global inhibitor that limits competitive, stochastically-activated clusters of Cdc42, and also as a lateral inhibitor of growth at hyphal tips that strongly influences the overall width of the hypha. In contrast, Rsr1-GTP, through Bud5 activity, is needed to efficiently nucleate single clusters of Cdc42 activity during hyphal emergence, and also contributes to the extremely narrow morphology of the hypha. Altogether, the data presented here suggest that Rsr1 cycling supports a conservative mechanism of polarization that optimizes the efficiency with which polarization occurs, which is required for the maintenance of polarized growth.

Fungal rhizomorphs are complex, multicellular, root-like organs formed through the aggregation, interlacing, and adhesion of millions of tip-growing hyphae. There has been very little research on the invasive mechanism utilized by rhizomorphs to penetrate compacted soils and woody substrates. Initial studies with Meruliporia incrassata, a wood-decay fungus that decomposes wooden components of buildings with an annual value of destruction estimated in millions of dollars, was aimed at inducing rhizomorphs in vitro. This attempt was not very successful, as only mycelial cords were produced. The pathogen Armillaria mellea was therefore chosen because it readily forms rhizomorphs in culture and serves as an excellent model for developmental studies. This dissertation presents findings from experiments designed to study (i) comparative features of rhizomorph anatomy in M. incrassata and A. mellea that support its invasive behavior; (ii) the adaptive growth response of rhizomorphs subjected to mechanical stress; (iii) the biochemical basis of turgor generation within rhizomorphs, and (iv) novel measurements of the forces exerted by growing rhizomorphs. Anatomical studies of rhizomorphs of A. mellea cultured in potato dextrose agar (PDA) revealed zones of hyphal tissues namely, an outer layer of peripheral hyphae, radial hyphae, longitudinal hyphae, and a central cavity. A. mellea rhizomorphs were observed to have faster growth than mycelia in PDA. We determined that increasing concentration of agar stimulated the production of more rhizomorphs, with those in media having higher concentration of agar extending faster with tapered tips. Turgor generation within A. mellea rhizomorphs was shown to be partially due to the accumulation of osmolytes. Erythritol and mannitol were identified using Gas Chromatography/Mass Spectrometry (GC/MS) and quantitatively determined to be the most dominant osmolytes that contribute to turgor generation. Osmometric studies revealed that substantial portion of turgor generated was used to exert pressure at the tip of the rhizomorphs during invasive growth. The varying amounts of force that these A. mellea rhizomorph tips exert were measured, using a sensitive strain gauge. Our experiments provide the first clear picture of the mechanical processes that allow rhizomorphs to function as migratory, exploratory and invasive organs in low-moisture and nutrient-poor environments that present substantial obstacles to fungal colonization.