Fusion PCR and gene targeting in Aspergillus nidulans

Szewczyk, Edyta; Nayak, Tania; Oakley, C. Elizabeth; Edgerton, Heather; Xiong, Yi; Taheri-Talesh, Naimeh; Osmani, Stephen A.; Oakley, Berl R.

doi:10.1038/nprot.2006.405

Cited by 676 publications

(715 citation statements)

References 13 publications

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“…Deletion strains were obtained by transformation (36) using the argB gene of A. nidulans as a selectable marker. Deletion cassettes were produced as described in Szewczyk (37). Strains with tagged gene variants were obtained using the pabaA1 gene of A. nidulans as a selectable marker.…”

Section: Methodsmentioning

confidence: 99%

Bacteria-induced natural product formation in the fungus Aspergillus nidulans requires Saga/Ada-mediated histone acetylation

Nützmann

Reyes-Domínguez

Scherlach

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

303

269

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Sequence analyses of fungal genomes have revealed that the potential of fungi to produce secondary metabolites is greatly underestimated. In fact, most gene clusters coding for the biosynthesis of antibiotics, toxins, or pigments are silent under standard laboratory conditions. Hence, it is one of the major challenges in microbiology to uncover the mechanisms required for pathway activation. Recently, we discovered that intimate physical interaction of the important model fungus Aspergillus nidulans with the soil-dwelling bacterium Streptomyces rapamycinicus specifically activated silent fungal secondary metabolism genes, resulting in the production of the archetypal polyketide orsellinic acid and its derivatives. Here, we report that the streptomycete triggers modification of fungal histones. Deletion analysis of 36 of 40 acetyltransferases, including histone acetyltransferases (HATs) of A. nidulans, demonstrated that the Saga/Ada complex containing the HAT GcnE and the AdaB protein is required for induction of the orsellinic acid gene cluster by the bacterium. We also showed that Saga/Ada plays a major role for specific induction of other biosynthesis gene clusters, such as sterigmatocystin, terrequinone, and penicillin. Chromatin immunoprecipitation showed that the Saga/Ada-dependent increase of histone 3 acetylation at lysine 9 and 14 occurs during interaction of fungus and bacterium. Furthermore, the production of secondary metabolites in A. nidulans is accompanied by a global increase in H3K14 acetylation. Increased H3K9 acetylation, however, was only found within gene clusters. This report provides previously undescribed evidence of Saga/Ada dependent histone acetylation triggered by prokaryotes.

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Section: Methodsmentioning

confidence: 99%

Bacteria-induced natural product formation in the fungus Aspergillus nidulans requires Saga/Ada-mediated histone acetylation

Nützmann

Reyes-Domínguez

Scherlach

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

303

269

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“…SECC was tagged at its C terminus by using fusion PCR to generate an appropriate transforming fragment Szewczyk et al, 2006). The A. fumigatus pyroA gene (AfpyroA) was used as a selectable marker.…”

Section: Fusion Polymerase Chain Reaction (Pcr) and Strain Constructionmentioning

confidence: 99%

“…Other primers were used for both GFP and mCherry fusions. PCR enzymes and conditions were as described by Szewczyk et al (2006). The A. fumigatus pyrG gene (AfpyrG) was used as a selectable marker.…”

Section: Fusion Polymerase Chain Reaction (Pcr) and Strain Constructionmentioning

confidence: 99%

The Tip Growth Apparatus ofAspergillus nidulans

Taheri-Talesh

Horio

Araújo‐Bazán

et al. 2008

MBoC

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271

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Hyphal tip growth in fungi is important because of the economic and medical importance of fungi, and because it may be a useful model for polarized growth in other organisms. We have investigated the central questions of the roles of cytoskeletal elements and of the precise sites of exocytosis and endocytosis at the growing hyphal tip by using the model fungus Aspergillus nidulans. Time-lapse imaging of fluorescent fusion proteins reveals a remarkably dynamic, but highly structured, tip growth apparatus. Live imaging of SYNA, a synaptobrevin homologue, and SECC, an exocyst component, reveals that vesicles accumulate in the Spitzenkö rper (apical body) and fuse with the plasma membrane at the extreme apex of the hypha. SYNA is recycled from the plasma membrane by endocytosis at a collar of endocytic patches, 1-2 m behind the apex of the hypha, that moves forward as the tip grows. Exocytosis and endocytosis are thus spatially coupled. Inhibitor studies, in combination with observations of fluorescent fusion proteins, reveal that actin functions in exocytosis and endocytosis at the tip and in holding the tip growth apparatus together. Microtubules are important for delivering vesicles to the tip area and for holding the tip growth apparatus in position. INTRODUCTIONPolarized cell growth occurs in most eukaryotic phyla, and it includes a plethora of important phenomena, such as neuronal growth cone extension in animals and pollen tube extension in vascular plants. It is particularly important in filamentous fungi where nearly all growth occurs by hyphal tip extension (reviewed by Momany, 2002). Given that some filamentous fungi are important fermentation organisms, the growth of which is of considerable economic importance, whereas others are serious plant, animal, and human pathogens, there is considerable interest in the mechanisms of tip growth in these organisms.A great deal of progress has been made in understanding fungal tip growth (summarized by Harris et al., 2005; Steinberg, 2007a,b;Riquelme et al., 2007), but key questions remain unanswered. There is general agreement that fungal tip growth involves the synthesis of cell wall components in the cell body, the incorporation of these components into vesicles, the transport of these vesicles to the cell tip, the fusion of these vesicles with the plasma membrane in the area of the cell tip (exocytosis) to release their contents, and the cross-linking of the components after release. It is clear that both microtubules and actin microfilaments play important roles in fungal tip growth, but their exact functions are not yet defined. The exact sites of exocytosis and endocytosis also remain to be determined. The positions of the site(s) of exocytosis are particularly important because fungal walls are relatively stiff structures, and once they have formed the shape of the hypha is established. Hyphal shape is thus determined to a very significant extent by where the wall precursors are released from the cytoplasm, i.e., by the positioning of the site(s) of exocyto...

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“…Growth of mycelium on cover slips for fluorescence confocal microscopy followed the method of Osmani et al 32 except that Novozyme Vinoflow FCE, prepared as described by Szewczyk et al, 33 was used to digest the cell walls to allow entry of antibodies. In these experiments the carbon source was 0.5% w/v quinic acid, 0.05% w/w glucose.…”

Section: Molecular Biology and Biochemistrymentioning

confidence: 99%

The transcription repressor NmrA is subject to proteolysis by three Aspergillus nidulans proteases

et al. 2010

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The role of specific cleavage of transcription repressor proteins by proteases and how this may be related to the emerging theme of dinucleotides as cellular signaling molecules is poorly characterized. The transcription repressor NmrA of Aspergillus nidulans discriminates between oxidized and reduced dinucleotides, however, dinucleotide binding has no effect on its interaction with the zinc finger in the transcription activator AreA. Protease activity in A. nidulans was assayed using NmrA as the substrate, and was absent in mycelium grown under nitrogen sufficient conditions but abundant in mycelium starved of nitrogen. One of the proteases was purified and identified as the protein Q5BAR4 encoded by the gene AN2366.2. Fluorescence confocal microscopy showed that the nuclear levels of NmrA were reduced approximately 38% when mycelium was grown on nitrate compared to ammonium and absent when starved of nitrogen. Proteolysis of NmrA occurred in an ordered manner by preferential digestion within a C-terminal surface exposed loop and subsequent digestion at other sites. NmrA digested at the C-terminal site was unable to bind to the AreA zinc finger. These data reveal a potential new layer of control of nitrogen metabolite repression by the ordered proteolytic cleavage of NmrA. NmrA digested at the C-terminal site retained the ability to bind NAD 1 and showed a resistance to further digestion that was enhanced by the presence of NAD 1 . This is the first time that an effect of dinucleotide binding to NmrA has been demonstrated.

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Fusion PCR and gene targeting in Aspergillus nidulans

Cited by 676 publications

References 13 publications

Bacteria-induced natural product formation in the fungus Aspergillus nidulans requires Saga/Ada-mediated histone acetylation

Bacteria-induced natural product formation in the fungus Aspergillus nidulans requires Saga/Ada-mediated histone acetylation

The Tip Growth Apparatus ofAspergillus nidulans

The transcription repressor NmrA is subject to proteolysis by three Aspergillus nidulans proteases

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