Yainitza Hernandez-Rodriguez

Hortaea werneckii is a halotolerant black yeast commonly found in hypersaline environments. This yeast is also the causative agent of tinea nigra, a superficial mycosis of the palm of the hand and soles of the feet of humans. In addition to their remarkable halotolerance, this black yeast exhibits an unconventional cell division cycle, alternating between fission and budding cell division. Cell density and the salt concentration in their environment regulate which cell division cycle H. werneckii uses. Although H. werneckii have been extensively studied due to their unique physiology and cell biology, deciphering the underlying mechanisms behind these remarkable phenotypes has been limited due to the lack of genetic tools available. Here, we report a new ectopic integration protocol for H. werneckii using polyethylene glycol-CaCl2 mediated protoplast transformation. This approach relies on a drug (hygromycin B) resistance gene to select for successful integration of the genetic construct. The same construct was used to express cytosolic green fluorescent protein. Finally, we developed a marker-free CRISPR/Cas9 protocol for targeted gene deletion using the melanin synthesis pathway as a visual reporter of successful transformation. These transformation strategies will allow testing hypotheses related to H. werneckii cell biology and physiology.IMPORTANCEHortaea werneckii is a remarkable yeast capable of growing in high salt concentration, and its cell division cycle alternates between fission-like and budding. For these unique attributes, H. werneckii has gathered interest in research programs studying extremophile fungi and cell division. Most of our understanding of H. werneckii biology comes from genomic analyses, the usage of drugs to target a particular pathway, or the heterologous expression of its genes in S. cerevisiae. Nonetheless, H. werneckii has remained genetically intractable. Here, we report on two strategies to transform H. werneckii: ectopic integration of a plasmid and gene deletion using CRISPR/Cas9. These approaches will be fundamental to expanding the experimental techniques available to study H. werneckii, including live-cell imaging of cellular processes and reverse genetic approaches.

Septins are important components of the cytoskeleton that are highly conserved in eukaryotes and play major roles in cytokinesis, patterning, and many developmental processes. Septins form heteropolymers which assemble into higher-order structures including rings, filaments, and gauzes. In contrast to actin filaments and microtubules, the molecular mechanism by which septins assemble is not well-understood. Here, we report that in the filamentous fungus Aspergillus nidulans, four core septins form heteropolymeric complexes. AspE, a fifth septin lacking in unicellular yeasts, interacts with only one of the core septins, and only during multicellular growth. AspE is required for proper localization of three of the core septins, and requires this same subset of core septins for its own unique cortical localization. The ΔaspE mutant lacks developmentally-specific septin higher-order structures and shows reduced spore production and slow growth with low temperatures and osmotic stress. Our results show that at least two distinct septin heteropolymer populations co-exist in A. nidulans, and that while AspE is not a subunit of either heteropolymer, it is required for assembly of septin higher-order structures found in multicellular development.

► There are no fungal septin residues that are universally modified and only a few pairs that are identically modified. ► Posttranslational modifications of septins appear to be evolutionarily recent. ► G domain posttranslational modifications cluster near the G interface, where they probably modulate heteropolymer formation. ► N-terminal and C-terminal modifications are scattered, where they probably modulate unique higher-order structures. Septins are cytoskeletal elements that contain a highly conserved canonical G domain flanked by more divergent N- and C-termini. Septin monomers form heteropolymers that in turn associate into a variety of higher-order structures. SUMOylation, acetylation and phosphorylation of septins have all been reported; however, there are no examples of residues that are universally modified suggesting that posttranslational modifications of septins evolved relatively recently. Within the conserved G domain, posttranslational modifications cluster in regions near the G interface, consistent with roles in modulating heteropolymer assembly. Within the highly diverged N- and C-termini, posttranslational modifications are scattered randomly, consistent with roles in modulating assembly of higher-order structures that are unique to individual organisms.

In yeast, septins form rings at the mother-bud neck and function as diffusion barriers. In animals, septins form filaments that can colocalize with other cytoskeletal elements. In the filamentous fungus Aspergillus nidulans there are five septin genes, aspA (an ortholog of Saccharomyces cerevisiae CDC11), aspB (an ortholog of S. cerevisiae CDC3), aspC (an ortholog of S. cerevisiae CDC12), aspD (an ortholog of S. cerevisiae CDC10), and aspE (found only in filamentous fungi). The aspB gene was previously reported to be the most highly expressed Aspergillus nidulans septin and to be essential. Using improved gene targeting techniques, we found that deletion of aspB is not lethal but results in delayed septation, increased emergence of germ tubes and branches, and greatly reduced conidiation. We also found that AspB-green fluorescent protein (GFP) localizes as rings and collars at septa, branches, and emerging layers of the conidiophore and as bars and filaments in conidia and hyphae. Bars are found in dormant and isotropically expanding conidia and in subapical nongrowing regions of hyphae and display fast movements. Filaments form as the germ tube emerges, localize to hyphal and branch tips, and display slower movements. All visible AspB-GFP structures are retained in Delta aspD and lost in Delta aspA and Delta aspC strains. Interestingly, in the Delta aspE mutant, AspB-GFP rings, bars, and filaments are visible in early growth, but AspB-GFP rods and filaments disappear after septum formation. AspE orthologs are only found in filamentous fungi, suggesting that this class of septins might be required for stability of septin bars and filaments in highly polar cells.

In addition to phialidic conidia (PC), A. terreus produces accessory conidia (AC) both in vitro and in vivo. AC are distinct from PC in cell surface architecture, with the AC surfaces displaying more β-glucan, a molecule that can be a trigger for the induction of inflammatory responses. The present study follows β-glucan cell surface presentation throughout the course of germination of both types of conidia, and analyzes the differential capacity of AC and PC to elicit immune responses. Results show that AC display early, increased dectin-1 labeling on their cell surfaces compared to PC, and this differential dectin-1 labeling is sustained on the cell surface from the time of breaking dormancy through early germ tube emergence. Mouse alveolar macrophages showed a stronger inflammatory cytokine/chemokine response when challenged with AC than with PC in both ex vivo and in vivo experiments, correlating with the greater exposure of β-glucan exhibited by AC. Further, histopathologic staining of the lungs from mice challenged with AC demonstrated heightened cell recruitment and increased inflammatory response compared to the lungs of mice challenged with PC. Our study also demonstrates that AC are multinucleate structures with the ability to germinate rapidly, polarizing in multiple directions and producing several hyphal extensions. We present evidence that A. terreus AC are phenotypically distinct from PC and can be potent activators of the innate immune mechanism thus possibly playing a role in this organism's pathogenesis.

Septins are cytoskeletal proteins found in fungi, animals, and microsporidia, where they form multiseptin complexes that act as scaffolds recruiting and organizing other proteins to ensure normal cell division and development. Here we characterize the septins AspA and AspC in the multicellular, filamentous fungus A spergillus nidulans . Mutants with deletions of aspA , aspC , or both aspA and aspC show early and increased germ tube and branch emergence, abnormal septation, and disorganized conidiophores. Strains in which the native aspA has been replaced with a single copy of aspA-GFP driven by the native septin promoter or in which aspC has been replaced with a single copy of aspC-GFP driven by the native promoter show wild-type phenotypes. AspA-GFP and AspC-GFP show identical localization patterns as discrete spots or bars in dormant and expanding conidia, as rings at forming septa and at the bases of emerging germ tubes and branches, and as punctate spots and filaments in the cytoplasm and at the cell cortex. In conidiophores, AspA-GFP and AspC-GFP localize as diffuse bands or rings at the bases of emerging layers and conidial chains and as discrete spots or bars in newly formed conidia. AspA-GFP forms abnormal structures in ΔaspC strains while AspC-GFP does not localize in ΔaspA strains. Our results suggest that AspA and AspC interact with each other and are important for normal development, especially for preventing the inappropriate emergence of germ tubes and branches. This is the first report of a septin limiting the emergence of new growth foci in any organism.