Functional Characterization of Aspergillus nidulans Homologues of Saccharomyces cerevisiae Spa2 and Bud6
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The importance of polarized growth for fungi has elicited significant effort directed at better understanding underlying mechanisms of polarization, with a focus on yeast systems. At sites of tip growth, multiple protein complexes assemble and coordinate to ensure that incoming building material reaches the appropriate destination sites, and polarized growth is maintained. One of these complexes is the polarisome that consists of Spa2, Bud6, Pea2, and Bni1 in Saccharomyces cerevisiae. Filamentous hyphae differ in their development and life style from yeasts and likely regulate polarized growth in a different way. This is expected to reflect on the composition and presence of protein complexes that assemble at the hyphal tip. In this study we searched for polarisome homologues in the model filamentous fungus Aspergillus nidulans and characterized the S. cerevisiae Spa2 and Bud6 homologues, SpaA and BudA. Compared to the S. cerevisiae Spa2, SpaA lacks domain II but has three additional domains that are conserved within filamentous fungi. Gene replacement strains and localization studies show that SpaA functions exclusively at the hyphal tip, while BudA functions at sites of septum formation and possibly at hyphal tips. We show that SpaA is not required for the assembly or maintenance of the Spitzenkörper. We propose that the core function of the polarisome in polarized growth is maintained but with different contributions of polarisome components to the process.Keywords:
Aspergillus nidulans
Filamentous fungus
Fungal protein
Filamentous fungi are widely used in the production of a variety of industrially relevant enzymes and proteins as they have the unique ability to secrete tremendous amounts of proteins. However, the secretory pathways in filamentous fungi are not completely understood. Here, we investigated the role of a mutation in the POlarity Defective (podB) gene on growth, protein secretion, and cell wall organization in Aspergillus nidulans using a temperature sensitive (Ts) mutant. At restrictive temperature, the mutation resulted in lack of biomass accumulation, but led to a significant increase in specific protein productivity. Proteomic analysis of the secretome showed that the relative abundance of 584 (out of 747 identified) proteins was altered due to the mutation. Of these, 517 were secreted at higher levels. Other phenotypic differences observed in the mutant include up-regulation of unfolded protein response (UPR), deformation of Golgi apparatus and uneven cell wall thickness. Furthermore, proteomic analysis of cell wall components in the mutant revealed the presence of intracellular proteins in higher abundance accompanied by lower levels of most cell wall proteins. Taken together, results from this study suggest the importance of PodB as a target when engineering fungal strains for enhanced secretion of valuable biomolecules.
Aspergillus nidulans
Secretory protein
Filamentous fungus
Fungal protein
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Fungal hydrophobins are secreted proteins that self-assemble at hydrophobic:hydrophilic interfaces. They are essential for a variety of processes in the fungal life cycle, including mediating interactions with surfaces and infection of hosts. The fungus Magnaporthe oryzae, the causative agent of rice blast, relies on the unique properties of hydrophobins to infect cultivated rice as well as over 50 different grass species. The hydrophobin MPG1 is highly expressed during rice blast pathogenesis and has been implicated during host infection. Here we report the backbone and sidechain assignments for the class I hydrophobin MPG1 from the rice blast fungus Magna- porthe oryzae.
Hydrophobin
Fungal protein
Filamentous fungus
Magnaporthe
Appressorium
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SUMMARY: The process of regeneration of mycelial protoplasts from Aspergillus nidulans wild-type and strain P76 has been investigated. In liquid media two patterns of regeneration were observed. In the first, the protoplasts produced chains of yeastlike cells and eventually the terminal cell produced a hypha. In the second wall 'shells' were formed into which the cytoplasm migrated. It is suggested that the mechanism of regeneration reflects the site of origin of the protoplast from the parent hypha.
Protoplast
Aspergillus nidulans
Strain (injury)
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Aspergillus nidulans
Cladosporium
Filamentous fungus
Fungal protein
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The V10 deteriorated variant of Aspergillus nidulans has hyphae, metulae, phialides and conidia with abnormal nuclear distributions.The alterations observed were: increase in the number of nuclei in hyphae, metulae and phialides, presence of anucleate, uninucleate and multinucleate conidia, abnormal vegetative growth and defective conidiation.When 0.5 M NaCl was added to the medium, an increase in the number of conidia was observed but their morphology and number of nuclei were not modified.The gene responsible for these alterations was named anuA1.The anuA1 gene is located on linkage group VII and is possibly involved in nuclear migration to hyphae, metulae, phialides and conidia.
Aspergillus nidulans
Conidiation
Multinucleate
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Hydrophobin
Magnaporthe
Filamentous fungus
Fungal protein
Appressorium
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We have studied compensatory evolution in a fludioxonil resistant mutant of the filamentous fungus Aspergillus nidulans. In an evolution experiment lasting for 27 weeks (about 3000 cell cycles) 35 parallel strains of this mutant evolved in three different environmental conditions. Our results show a severe cost of resistance (56%) in the absence of fludioxonil and in all conditions the mutant strain was able to restore fitness without loss of the resistance. In several cases, the evolved strain reached a higher fitness than the original sensitive ancestor. Fitness compensation occurred in one, two or three discrete steps. Genetic analysis of crosses between different evolved strains and between evolved and ancestral strains revealed interaction between compensatory mutations and provided information on the number of loci involved in fitness compensation. In addition, we discuss the opportunities for the experimental study of evolutionary processes provided by the filamentous fungus A. nidulans.
Aspergillus nidulans
Filamentous fungus
Fludioxonil
Experimental Evolution
Strain (injury)
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Class III chitin synthases play important roles in tip growth and conidiation in many filamentous fungi. However, little is known about their functions in those processes. To address these issues, we characterized the deletion mutant of a class III chitin synthase-encoding gene of Aspergillus nidulans, chsB, and investigated ChsB localization in the hyphae and conidiophores. Multilayered cell walls and intrahyphal hyphae were observed in the hyphae of the chsB deletion mutant, and wavy septa were also occasionally observed. ChsB tagged with FLAG or enhanced green fluorescent protein (EGFP) localized mainly at the tips of germ tubes, hyphal tips, and forming septa during hyphal growth. EGFP-ChsB predominantly localized at polarized growth sites and between vesicles and metulae, between metulae and phialides, and between phalides and conidia in asexual development. These results strongly suggest that ChsB functions in the formation of normal cell walls of hyphae, as well as in conidiophore and conidia development in A. nidulans.
Aspergillus nidulans
Chitin synthase
Conidiation
Tip growth
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The analysis of the transverse septa of the mycelium of an ectendomycorrhizal fungus of the pine (strain MrgX 1) using a transmission electron microscope has shown that the septa contain simple pores, which indicates that the fungus belongs to the <i>Ascomycetes</i>.
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Cell division (cytokinesis) in filamentous fungi involves inward growth of the plasma membrane at sites of crosswall (septum) formation, under the guidance of a contractile actomyosin ring (CAR). The process involves the coordinated activities of multiple proteins, most of which are poorly characterized or wholly undiscovered. A forward‐genetics approach to identifying proteins with unrecognized roles in cell division has been to artificially mutagenize cells and isolate viable progeny defective in septum formation (septation), followed by identification of mutation sites at the molecular level. Prior work in this laboratory has localized a classic temperature‐sensitive septation mutation, termed sepG1 , in the model fungus Aspergillus nidulans to a gene locus predicted to encode an IQGAP homologue. Reduced expression of the wild type gene under the regulatable AlcA promoter blocks septation, and a GFP‐tagged version of the wild type protein localizes to sites where septa are forming. We have also succeeded in GFP‐tagging the protein encoded by the mutant sepG1 allele and have demonstrated that it too localizes to septation sites, even under restrictive conditions that prevent completion of septation, indicating that the sepG1 defect is not in protein targeting. Multiple attempts using Mendelian methods to create double‐mutant strains expressing both GFP‐tagged SepG1 protein plus RFP‐tagged versions of actin or myosin have not been successful, preventing investigation of whether the SepG1 protein associates normally with the CAR. Therefore, we have generated riboflavin‐auxotrophic strains containing untagged sepG1 mutant alleles along with RFP‐tagged wild‐type alleles of actin or myosin, with the goal of tagging the sepG1 allele in these strains via direct genetic transformation. We have constructed a plasmid‐based cassette containing sequences that, after integration, will allow the simultaneous expression of GFP‐tagged SepG1 protein in the same strains that are also expressing RFP‐tagged actin or myosin. Support or Funding Information National Science Foundation Grant 1615192 (Loretta Jackson‐Hayes, PI). This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
Aspergillus nidulans
Wild type
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