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Two Components of a velvet-Like Complex Control Hyphal Morphogenesis, Conidiophore Development, and Penicillin Biosynthesis in Penicillium chrysogenum

Birgit Hoff, Jens Kamerewerd, Claudia Sigl, Rudolf Mitterbauer, Ivo Zadra, Hubert Kürnsteiner, Ulrich Kück
Birgit Hoff
1Christian Doppler Laboratory for Fungal Biotechnology, Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, Universitätsstraße 150, 44780 Bochum, Germany
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Jens Kamerewerd
1Christian Doppler Laboratory for Fungal Biotechnology, Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, Universitätsstraße 150, 44780 Bochum, Germany
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Claudia Sigl
2Sandoz GmbH, Biochemiestraße 10, 6250 Kundl, Austria
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Rudolf Mitterbauer
2Sandoz GmbH, Biochemiestraße 10, 6250 Kundl, Austria
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Ivo Zadra
2Sandoz GmbH, Biochemiestraße 10, 6250 Kundl, Austria
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Hubert Kürnsteiner
2Sandoz GmbH, Biochemiestraße 10, 6250 Kundl, Austria
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Ulrich Kück
1Christian Doppler Laboratory for Fungal Biotechnology, Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, Universitätsstraße 150, 44780 Bochum, Germany
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  • For correspondence: ulrich.kueck@rub.de
DOI: 10.1128/EC.00077-10
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  • Fig. 1.
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    Fig. 1.

    Microarray analysis of the ΔPcvelA strain compared to the ΔPcku70 strain. (A) Venn diagrams of up- and downregulated genes from three time points (48, 60, and 96 h). The dark-blue sections contain genes that are significantly differently expressed during all times measured. Genes that showed at least a 2-fold up- or downregulation in the array hybridization were considered. (B) Functional categories of genes regulated by PcVelA. All genes that were up- or downregulated at a single time point in the ΔPcvelA strain compared to the ΔPcku70 strain were considered.

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    Fig. 2.

    Microarray analysis of selected Pcku70- and PcvelA-dependent genes. Heat maps of developmental genes (A) and genes of secondary and amino sugar metabolisms (B) are shown. Red, upregulation in the ΔPcvelA mutant compared to the ΔPcku70 strain; green, downregulation in the ΔPcvelA mutant compared to the ΔPcku70 strain. The color of each square represents the log2-fold change in expression at the given time point.

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    Fig. 3.

    Penicillin biosynthesis of knockout mutants. Transcript levels of pcbC and penDE genes in the ΔPclaeA (A) and ΔPcvelA (B) deletion strains and their corresponding complemented strains, which are indicated by “T.” Quantification of autoradiograms as shown was done with Scion Image. The gpdA transcript level served as a reference for all transcripts from individual strains, and the level in the ΔPcku70 strain was set to 100%. (C) HPLC analysis for the quantification of the penicillin V titer in recipient and knockout strains after 72 h of growth in submerged-liquid shaking cultures. Mean values and standard deviations are derived from three independent experiments. (D) Penicillin bioassays with S. aureus as a Gram-positive indicator bacterium.

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    Fig. 4.

    Morphogenesis of the ΔPcvelA and ΔPclaeA strains as well as the corresponding recipients and complemented strains. (A) Quantification of light- and dark-dependent conidial formation in strains as indicated. All strains were grown for 168 h under constant light or dark conditions. The experiment was performed in triplicate; bars show standard deviations. (B) Scanning electron microscopy of conidiophores from the wild-type P2niaD18 and the ΔPclaeA mutant strains. While P2niaD18 produces long chains of conidia (indicated by arrows), the ΔPclaeA strain consistently forms only a single conidium from a terminal vesicle of each phialide (indicated by arrows). Scale bars are valid for either both top or three bottom figures. (C) PcvelA-dependent hyphal morphology after 72 h of cultivation in surface liquid culture. White arrows indicate dichotomous hyphal branching sites. (D) Calcofluor white staining of hyphae from strains as indicated. Hyphae were stained after 48, 72, and 96 h of growth in surface-liquid cultures. (E) PcvelA-dependent pellet formation after 72, 120, and 168 h in submerged-liquid shaking cultures. (F) PclaeA-dependent hyphal morphology after 72 h of cultivation in surface-liquid culture. White arrows indicate dichotomous hyphal branching sites.

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    Fig. 5.

    Bimolecular fluorescence complementation studies. (A) Control to confirm the nuclear localization of two interacting velvet-like complex components by DAPI staining and demonstration that a strain producing only the two split EYFPs lacks any fluorescence. (B) Protein-protein interactions of components of the velvet-like protein complex in P. chrysogenum as indicated. The fusions of each gene pair with fragments encoding the N or C terminus of EYFP are given above each image set. Micrographs illustrate DIC microscopy, DAPI, or EYFP fluorescence and the merged image, as indicated.

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    Fig. 6.

    Heterologous complementation experiments with P. chrysogenum mutants using homologues from F. fujikuroi. Penicillin bioassays of complemented strains obtained after transformation of the ΔPcvelA strain with the homologous gene Ffvel1 (A) or the ΔPclaeA strain with Fflae1 (B). As reference, the recipient ΔPcku70 strain as well as the knockout ΔPcvelA and ΔPclaeA mutant strains are shown. From four different complemented strains, two are given as examples. In all cases, S. aureus served as a Gram-positive indicator. (C) Photographs of the recipient ΔPcku70 strain, the ΔPclaeA knockout mutant, and the complemented ΔPclaeA::Fflae1 T2 strain. Green pigmentation indicates proper conidial formation.

Tables

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  • Table 1.

    Fungal strains used in this study

    StrainGenotypeaReference or source
    P2niaD18 niaD − 33
    ΔPcku70 strainΔPcku70::nat1niaD− 31
    ΔPcvelA strainΔPcvelA::ble ΔPcku70::nat1niaD− 31
    ΔPcvelA::PcvelA strainΔPcvelA::ble ΔPcku70::nat1 PpgkA::PcvelA::TpgkAptrAniaD−This study
    ΔPclaeA strainΔPclaeA::ble ΔPcku70::nat1niaD−This study
    ΔPclaeA::PclaeA strainΔPclaeA::ble ΔPcku70::nat1 PPclaeA::Pclae::TPclaeAptrAniaD−This study
    ΔPclaeA::Fflae1 strainΔPclaeA::ble ΔPcku70::nat1 Pgpd::Fflae1::eyfpN::TtrpCptrAniaD−This study
    P2Bi-1Pgpd::PcvelA::eyfpN::TtrpC Pgpd::PcvelB::eyfpC::TtrpC PtrpC::nat1niaD−This study
    P2Bi-2Pgpd::PcvelA::eyfpN::TtrpC Pgpd::PclaeA::eyfpC::TtrpC PtrpC::nat1niaD−This study
    P2Bi-3Pgpd::PcvelA::eyfpN::TtrpC Pgpd::PckapA::eyfpC::TtrpC PtrpC::nat1niaD−This study
    P2Bi-4Pgpd::PcvelA::eyfpN::TtrpC PtrpC::nat1niaD−This study
    P2Bi-5Pgpd::PclaeA::eyfpC::TtrpC PtrpC::nat1niaD−This study
    P2Bi-6Pgpd::PcvelB::eyfpC::TtrpC PtrpC::nat1niaD−This study
    P2Bi-7Pgpd::PckapA::eyfpC::TtrpC PtrpC::nat1niaD−This study
    P2Bi-8Pgpd::eyfpC::TtrpC Pgpd::eyfpN::TtrpC PtrpC::nat1niaD−This study
    • ↵a eyfp N, eyfp N terminus; eyfpC, eyfp C terminus.

Additional Files

  • Figures
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  • Supplemental material

    Files in this Data Supplement:

    • Supplemental file 1 - Differentially expressed genes in the ΔPcvelA deletion mutant compared to those in the ΔPcku70 recipient strain.
      Zipped MS Word document, 12K.
    • Supplemental file 2 - Oligonucleotides used in this work.
      MS Word document, 48K.
    • Supplemental file 3 - lasmid constructs employed in this study.
      MS Word document, 37K.
    • Supplemental file 4 - Comparison of microarray and quantitative real-time PCR data.
      Zipped PDF file, 15K.
    • Supplemental file 5 - Multiple sequence alignment of the P. chrysogenum velvet-like protein PcvelB with its homologue VelB from A. nidulans.
      Zipped PDF file, 42K.
    • Supplemental file 6 - Fluorescence microscopic analysis of different control strains used in the BiFC approach.
      Zipped PDF file, 53K.
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Two Components of a velvet-Like Complex Control Hyphal Morphogenesis, Conidiophore Development, and Penicillin Biosynthesis in Penicillium chrysogenum
Birgit Hoff, Jens Kamerewerd, Claudia Sigl, Rudolf Mitterbauer, Ivo Zadra, Hubert Kürnsteiner, Ulrich Kück
Eukaryotic Cell Aug 2010, 9 (8) 1236-1250; DOI: 10.1128/EC.00077-10

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Two Components of a velvet-Like Complex Control Hyphal Morphogenesis, Conidiophore Development, and Penicillin Biosynthesis in Penicillium chrysogenum
Birgit Hoff, Jens Kamerewerd, Claudia Sigl, Rudolf Mitterbauer, Ivo Zadra, Hubert Kürnsteiner, Ulrich Kück
Eukaryotic Cell Aug 2010, 9 (8) 1236-1250; DOI: 10.1128/EC.00077-10
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KEYWORDS

Fungal Proteins
Hyphae
Morphogenesis
Penicillins
Penicillium chrysogenum
Spores, Fungal

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