Development of a homologous transformation system for the human pathogenic fungus Aspergillus fumigatus based on the pyrG gene encoding orotidine 5′′-monophosphate decarboxylase

Weidner, Gerhard; d’Enfert, Christophe; Koch, Andreas; Mol, P. C.; Brakhage, Axel A.

doi:10.1007/s002940050350

Cited by 198 publications

(170 citation statements)

References 27 publications

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“…To eliminate this concern, we have cloned the A. fumigatus homologs of the A. nidulans riboB and pyroA genes to use as selectable markers. We have also used the previously cloned A. fumigatus pyrG gene (Weidner et al 1998) and we have constructed a vector that confers resistance to glufosinate (phosphinothricin).…”

Section: Resultsmentioning

confidence: 99%

A Versatile and Efficient Gene-Targeting System for Aspergillus nidulans

et al. 2006

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Aspergillus nidulans is an important experimental organism, and it is a model organism for the genus Aspergillus that includes serious pathogens as well as commercially important organisms. Gene targeting by homologous recombination during transformation is possible in A. nidulans, but the frequency of correct gene targeting is variable and often low. We have identified the A. nidulans homolog (nkuA) of the human KU70 gene that is essential for nonhomologous end joining of DNA in double-strand break repair. Deletion of nkuA (nkuAD) greatly reduces the frequency of nonhomologous integration of transforming DNA fragments, leading to dramatically improved gene targeting. We have also developed heterologous markers that are selectable in A. nidulans but do not direct integration at any site in the A. nidulans genome. In combination, nkuAD and the heterologous selectable markers make up a very efficient genetargeting system. In experiments involving scores of genes, 90% or more of the transformants carried a single insertion of the transforming DNA at the correct site. The system works with linear and circular transforming molecules and it works for tagging genes with fluorescent moieties, replacing genes, and replacing promoters. This system is efficient enough to make genomewide gene-targeting projects feasible.

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Section: Resultsmentioning

confidence: 99%

A Versatile and Efficient Gene-Targeting System for Aspergillus nidulans

et al. 2006

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“…fumigatus Strains-A derivate of the A. fumigatus clinical isolate D141 deficient in non-homologous end-joining (AfS35) was used (34). A. fumigatus gmtA was replaced by a p gpdA::ble/tk::trpC t cassette (35), using electroporation of conidia (36). Generation of the replacement cassette is described in the supplemental material and primers used are listed in , and phases were separated by centrifugation.…”

Section: Methodsmentioning

confidence: 99%

Biosynthesis of the Fungal Cell Wall Polysaccharide Galactomannan Requires Intraluminal GDP-mannose

Engel

Schmalhorst

Routier

2012

Journal of Biological Chemistry

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Background: Understanding the mechanisms of cell wall biogenesis is important for development of antifungal strategies. Results: Biosynthesis of the fungal polysaccharide galactomannan requires import of GDP-mannose into the Golgi apparatus. Conclusion: It differs from the biosynthesis of other fungal cell wall polysaccharides.Significance: This provides a new paradigm for cell wall polysaccharide biosynthesis.

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“…Plasmid pTPS17 was then obtained by subcloning a KspI-EcoRV fragment carrying the A. fumigatus pyrG gene (Weidner et al, 1998) into KspI\SmaI-digested pTPS13. Plasmid pTPS17 was used to transform protoplasts of A. nidulans strains (Osmani et al, 1987).…”

Section: Methodsmentioning

confidence: 99%

Trehalose is required for the acquisition of tolerance to a variety of stresses in the filamentous fungus Aspergillus nidulans The GenBank accession number for the sequence reported in this paper is AF043230.

et al. 2001

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Trehalose is a non-reducing disaccharide found at high concentrations in Aspergillus nidulans conidia and rapidly degraded upon induction of conidial germination. Furthermore, trehalose is accumulated in response to a heat shock or to an oxidative shock. The authors have characterized the A. nidulans tpsA gene encoding trehalose-6-phosphate synthase, which catalyses the first step in trehalose biosynthesis. Expression of tpsA in a Saccharomyces cerevisiae tps1 mutant revealed that the tpsA gene product is a functional equivalent of the yeast Tps1 trehalose-6-phosphate synthase. The A. nidulans tpsA-null mutant does not produce trehalose during conidiation or in response to various stress conditions. While germlings of the tpsA mutant show an increased sensitivity to moderate stress conditions (growth at 45 SC or in the presence of 2 mM H 2 O 2 ), they display a response to severe stress (60 min at 50 SC or in the presence of 100 mM H 2 O 2 ) similar to that of wild-type germlings. Furthermore, conidia of the tpsA mutant show a rapid loss of viability upon storage. These results are consistent with a role of trehalose in the acquisition of stress tolerance. Inactivation of the tpsA gene also results in increased steady-state levels of sugar phosphates but does not prevent growth on rapidly metabolizable carbon sources (glucose, fructose) as seen in Saccharomyces cerevisiae. This suggests that trehalose 6-phosphate is a physiological inhibitor of hexokinase but that this control is not essential for proper glycolytic flux in A. nidulans. Interestingly, tpsA transcription is not induced in response to heat shock or during conidiation, indicating that trehalose accumulation is probably due to a post-translational activation process of the trehalose 6-phosphate synthase.

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Development of a homologous transformation system for the human pathogenic fungus Aspergillus fumigatus based on the pyrG gene encoding orotidine 5′′-monophosphate decarboxylase

Cited by 198 publications

References 27 publications

A Versatile and Efficient Gene-Targeting System for Aspergillus nidulans

A Versatile and Efficient Gene-Targeting System for Aspergillus nidulans

Biosynthesis of the Fungal Cell Wall Polysaccharide Galactomannan Requires Intraluminal GDP-mannose

Trehalose is required for the acquisition of tolerance to a variety of stresses in the filamentous fungus Aspergillus nidulans The GenBank accession number for the sequence reported in this paper is AF043230.

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