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The Septin AspB in Aspergillus nidulans Forms Bars and Filaments and Plays Roles in Growth Emergence and Conidiation

Yainitza Hernández-Rodríguez, Susan Hastings, Michelle Momany
Yainitza Hernández-Rodríguez
University of Georgia, Athens, Georgia, USA
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Susan Hastings
University of Georgia, Athens, Georgia, USA
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Michelle Momany
University of Georgia, Athens, Georgia, USA
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DOI: 10.1128/EC.05164-11
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  • Fig 1
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    Fig 1

    aspB is required for normal growth and morphology. (A) The ΔaspB mutant forms uninucleate and binucleate spores. Dormant spores from wt, aspB-gfp, and ΔaspB strains were stained with Hoechst 33342, and the numbers of nuclei were counted (n = 200). (B) The ΔaspB mutant breaks dormancy earlier than the wild-type strain. Spores were incubated for 4 h at 30°C and categorized as dormant (∼2.5 μm), isotropic (∼5 μm), and polar (presence of a germ tube) (n = 200). (C) The ΔaspB mutant forms multiple germ tubes. Spores were incubated for 6 h at 30°C, and the numbers of germ tubes were counted (n = 300). (D) The ΔaspB mutant delays septation. Spores were incubated for 11 h at 30°C, and the number of septa were counted (n = 200). (E) The ΔaspB mutant hyperbranches. Spores were incubated for 14 h at 30°C, and the numbers of branches per compartment delineated by two septa were counted (n = 200). (F) The ΔaspB mutant forms abnormal conidiophores. Spores were incubated in agar between coverslips for 2 days at 30°C. Conidiophores were categorized as normal if all layers were present and abnormal if layers were absent or aberrant (n = 200).

  • Fig 2
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    Fig 2

    The ΔaspB mutant shows a hyper-emergence of growth and abnormal conidiophores. (A) The ΔaspB mutant forms multiple germ tubes, whereas the wild-type strain forms one germ tube. (B) The ΔaspB mutant forms multiple abnormal and stunted branches per compartment, whereas the wild-type strain forms one branch per compartment. (C) The ΔaspB mutant forms disorganized conidiophores. An arrow denotes a new aerial hypha arising from a vesicle. The wild-type strain forms many conidia, and the conidiophores are organized with different layers (V, vesicle; P, phialide; M, metulae; Cc, conidial chain). Conditions for growth were as described in Fig. 1. Scale bar, 5 μm.

  • Fig 3
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    Fig 3

    The ΔaspB mutant shows clumped nuclei. (A) Wild-type nuclei positioned along hyphae. (B) The ΔaspB mutant results in clumped nuclei, particularly near branches. (C) The ΔaspB mutant shows clumped nuclei. The ΔaspB mutant showed more nuclei in 15 μm around the most basal branch than did the wild-type strain. To delineate the area for nuclear counts, the most basal branch was identified; 7.5 μm from the center of the branch to the left and right of the branch was measured (total area = 15 μm; n = 200). Spores were incubated for 14 to 16 h at 30°C, and nuclei were stained with Hoechst 33258. Scale bar, 5 μm.

  • Fig 4
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    Fig 4

    AspB forms rings, dots, bars, and filaments. The AspB-GFP strain was incubated at 30°C for 4 to 5 h to view isotropic growth, 6 to 7 h to view early germ tube emergence, 9 to 11 h to view hyphal elongation and septation, and 12 to 16 h to view branching. (A) AspB forms bars (*), rings (<), and dots (arrowhead) in dormant and germinating spores. The inset shows an enlarged view of a ring (scale bar, 0.5 μm). (B to D) AspB forms “X's” (#) in addition to bars, rings, and dots as conidia swell. The inset shows an enlarged view of an “X” (scale bar, 0.75 μm). AspB forms caps and collars as the germ tube emerges, bars that localize to conidia and filaments (arrows) that localize to tips. (E to G) AspB forms bars that localize subapically and filaments that localize to tips as the hypha extends. Cytoplasmic GFP fluorescence is excluded from nuclei (N). (H) AspB forms rings at septa. (I to R) AspB forms filaments in branching compartments, caps as the branch emerges, and dots at the tips of branches. Filaments localize to newly formed branches. (S) Filaments and bars localize to longer branches. (T) Top view of a branch cap. Scale bar, 5 μm.

  • Fig 5
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    Fig 5

    AspB forms bars, filaments, caps, and rings during conidiophore formation. (A) AspB-GFP localization at several stages of early conidiophore development. AspB-GFP forms bars (*) and filaments (arrow) in conidiophore stalks. AspB-GFP forms caps (C) in aerial hyphae. AspB-GFP caps are diffuse in swollen vesicles. (B) AspB-GFP remains localized at the phialide-conidium interface as conidia form. AspB-GFP forms rings (>) at the base of budding layers. (C) Conidial chains attached to conidiophores show AspB-GFP localization as dots and bars. (D) AspB-GFP forms bars on conidia freshly detached from conidiophores. Some bars looked “frayed” (X) at the ends. Spores were incubated in agar between coverslips for 1, 2, and 3 days at 30°C and viewed by confocal microscopy. Scale bar, 5 μm.

  • Fig 6
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    Fig 6

    Motion of AspB bars and filaments. (A) AspB-GFP bar (*) in a germinating conidium moves away from the cell periphery, oscillates in and out of the plane of focus, and breaks in two. “Frayed” ends (X) can be observed. (B) AspB-GFP filaments (arrow) show slower movement than bars at the hyphal tips. Cytoplasmic GFP fluorescence is excluded from nuclei. Time lapse fluorescence microscopy was used. Numbers represent time lapse frames from Movies S1 and S2 in the supplemental material. Frames are 5 to 10 s apart. Conditions for growth as in Fig. 4. Scale bar, 5 μm.

  • Fig 7
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    Fig 7

    AspB structures are lost in the absence of AspA and AspC and filaments and bars are lost in hyphae in the absence of AspE. (I) AspB-GFP structures in septin deletion backgrounds. (A) AspB-GFP structures. Rings (>), dots (arrowhead), bars (*) filaments (arrow), and at septa (S) are indicated. (B) All AspB-GFP structures are lost in the ΔaspA ΔaspC mutant. (C) All AspB-GFP structures are found in ΔaspD strains. (D) All AspB-GFP structures are found in early ΔaspE cells but are mostly seen as dots and/or “X's” in hyphae. Conditions for growth were as described in Fig. 4. Scale bar, 5 μm. (II) AspB-GFP structures found in septin deletion backgrounds. Spores were incubated for 10 h at 30°C, and AspB structures were classified as filament, bar, and spot (rings, dots, and X's) and cytoplasmic (n = 100). Scale bar, 5 μm.

  • Fig 8
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    Fig 8

    AspB bars are lost in conidiophores in the absence of AspA and AspC. (A) AspB-GFP bars (*) in conidiophores. (B) AspB-GFP bars are lost in conidiophores in the ΔaspA ΔaspC mutant. (C) AspB-GFP bars and dots (arrowhead) in the ΔaspD mutant. (D) AspB-GFP bars and dots in the ΔaspE mutant. Conditions for growth were as described as in Fig. 5. Scale bar, 5 μm.

  • Fig 9
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    Fig 9

    Model of septin interactions in Aspergillus nidulans. (Left panel) Order of septin subunits in S. cerevisiae (adapted with permission from reference 6; additional data are from reference 43). (Right panel) Postulated order of septin subunits in A. nidulans. The wide end of septin subunit represents the NC interface; the narrow end represents the G interface. Boldface text denotes a single heteropolymer septin rod. The lighter text represents neighboring rods. Arrows indicate that septin polymer can be extended. X's indicate that septin polymer cannot be extended. Dark shading of AspC indicates that it differs from its S. cerevisiae ortholog Cdc12 because it is unable to form homopolymers via its G interface. See the text for further details.

Tables

  • Figures
  • Additional Files
  • Table 1

    Strains and plasmids used in this study

    Strain or plasmidGenotypeaSource or referenceb
    pFNO3ga5-gfp AfpyrG kanFGSC (67)
    A1145 (TN02A7)pyrG89; pyroA4; nkuA::argB; riboB2FGSC (67)
    A1147 (TNO2A25)pyrG89; argB2; pabaB22nku::argB; riboB2FGSC (67)
    A850biA1; _argB::trpC_B; methG1; veA1; trpC801FGSC
    A773pyrG89; wA3; pyroA4FGSC
    ARL115pyrG89 aspB::aspB-gfp-AfpyrG; argB2; pabaB22; nku::argB; riboB2This study
    ARL144pyrG89 aspB::AfpyrG; pyroA4; nkuA::argB; riboB2This study
    ARL148pyrG89 aspD::AfpyrG; pyroA4; nkuA::argB; riboB2This study
    ARL157aspC::AfpyrG; pyrG89; pyroA438
    ARL162aspC::AfpyrG; aspA::argB2; pyrG89; pyroA4; biA1_argB::trpC_B; veA1; trpC80138
    AYR1pyrG89 aspB::AfpyrG; pyroA4; riboB2This study
    AYR6pyrG89 aspB::aspB-gfp-AfpyrG; argB2This study
    AYR10aspB::aspB-gfp-AfpyrG\\This study
    AYR20aspB::aspB-gfp-AfpyrG; aspA::argB2This study
    AYR21aspB::aspB-gfp-AfpyrG; aspA::argB2This study
    AYR22aspB::aspB-gfp-AfpyrG; aspA::argB2This study
    AYR23aspB::aspB-gfp-AfpyrG; aspC::AfpyrGThis study
    AYR24aspB::aspB-gfp-AfpyrG; aspC::AfpyrGThis study
    AYR25aspB::aspB-gfp-AfpyrG; aspC::AfpyrG; aspA::argB2This study
    AYR26aspB::aspB-gfp-AfpyrG; aspE::AfpyrGThis study
    AYR27aspB::aspB-gfp-AfpyrG; aspE::AfpyrGThis study
    AYR32aspB::AfpyrG; pyroA4; argB2This study
    AYR35aspB::aspB-gfp-AfpyrG; pyroA4This study
    AYR43aspD::AfpyrGThis study
    AYR45aspB::aspB-gfp-AfpyrG; nkuA::argB; aspD::AfpyrThis study
    AYR64aspB::aspB-gfp-AfpyrG; pyroA4; aspD::pyrGAfThis study
    AYR65aspB::aspB-gfp-AfpyrG; pyroA4; aspD::pyrGAfThis study
    ASH26aspA::argB2 pyrG89 wa3 argB::trpC_B methG1 pyroA438
    ASH41aspE::AfpyrG; riboB2This study
    • ↵a The symbol “\\” indicates haploids fused to make a diploid strain.

    • ↵b FGSC, Fungal Genetics Stock Center, Department of Microbiology, University of Kansas Medical Center (Kansas City, KS).

  • Table 2

    Primers for constructing aspB-gfp and ΔaspBstrains

    PrimerSequence (5′–′3)
    For aspB GFP tag
        AspB-GSP1CAGAAGGTGCAACCTGTTCAGGGGAACTTAC
        AspB-GSP2ACGAAGAGAGAATCCCTTCCTCTTTCCCTTTTC
        AspB-GFP1GGAAAGAGGAAGGGATTCTCTCTTCGTGGAGCTGGTGCAGGCGCTG
        AspB-GFP2CGGGGTTTCCGACTAAGCGTCTGTCTGTCTGAGAGGAGGCACTGATGCG
        AspB-GSP3ACAGACGCTTAGTCGGAAACCCCGACGGTC
        AspB-GSP4GATACTGAACGTTCTCATCGCCCGCAAGC
        AspB-SSP3GCGTCGATGCTAAGAATTAGCTTCCC
        AspB-SSP4CGAGATCCATGCTAGCGTCATAGTAC
    For aspB deletion
        AspB-Up-FCTGTTCAATTGGATACTGCCGAG
        AspB-Up-RGAAGATGGAGTCAGCAGCTGTATAGG
        AspB pyrG1GCCTATACAGCTGCTGACTCCATCTTCTGCCTCAAACAATGCTCTTCACCCTC
        AspB pyrG2GTGGAGAATCAAACGTAGAAGTTCCAATAAGTGTCTGAGAGGAGGCACTGATGCG
        AspB-Down-FCTTCTACGTTTGATTCTCCACG
        AspB-Down-RCTACAGGATGACACCCAGTCAG
        AspBKOck-upGGTCATTCCTGGTGTGACAGTACC
        pyrgAFcheck RvCAGAGCCCACAGAGCGCCTTGAG

Additional Files

  • Figures
  • Tables
  • Supplemental material

    Files in this Data Supplement:

    • Supplemental file 1 - AspB bars show rapid oscillating movements.
      AVI Movie file, 963K.
    • Supplemental file 2 - AspB filaments show slower gradual movements.
      AVI Movie file, 2.7MB.
    • Supplemental file 3 - Legends for supplemental movies 1 and 2.
      MS Word document, 15K.
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The Septin AspB in Aspergillus nidulans Forms Bars and Filaments and Plays Roles in Growth Emergence and Conidiation
Yainitza Hernández-Rodríguez, Susan Hastings, Michelle Momany
Eukaryotic Cell Feb 2012, 11 (3) 311-323; DOI: 10.1128/EC.05164-11

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The Septin AspB in Aspergillus nidulans Forms Bars and Filaments and Plays Roles in Growth Emergence and Conidiation
Yainitza Hernández-Rodríguez, Susan Hastings, Michelle Momany
Eukaryotic Cell Feb 2012, 11 (3) 311-323; DOI: 10.1128/EC.05164-11
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