High‐Throughput Gene Replacement in <i>Aspergillus fumigatus</i>

Zhao, Can; Fraczek, Marcin G.; Dineen, Lauren; Lebedinec, Ressa; Macheleidt, Juliane; Heinekamp, Thorsten; Delneri, Daniela; Bowyer, Paul; Brakhage, Axel A.; Bromley, Michael

doi:10.1002/cpmc.88

Cited by 45 publications

(41 citation statements)

References 18 publications

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“…Null mutants were generated in the A. fumigatus strain MFIG001 by directed replacement of the TF of interest with a hygromycin (hph) selectable marker as described in Zhao et al 57 and validated using the processes outlined in Furukawa et al 33 . Overexpression mutants of the transcription factor encoding the genes AFUB_082490 and AFUB_089440 were created following previously published protocols for overexpression cassette construction 58 , protoplast generation and transformation 59 .…”

Section: Methodsmentioning

confidence: 99%

Fungal oxylipins direct programmed developmental switches in filamentous fungi

et al. 2020

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Filamentous fungi differentiate along complex developmental programs directed by abiotic and biotic signals. Currently, intrinsic signals that govern fungal development remain largely unknown. Here we show that an endogenously produced and secreted fungal oxylipin, 5,8-diHODE, induces fungal cellular differentiation, including lateral branching in pathogenic Aspergillus fumigatus and Aspergillus flavus, and appressorium formation in the rice blast pathogen Magnaporthe grisea. The Aspergillus branching response is specific to a subset of oxylipins and is signaled through G-protein coupled receptors. RNA-Seq profiling shows differential expression of many transcription factors in response to 5,8-diHODE. Screening of null mutants of 33 of those transcription factors identifies three transcriptional regulators that appear to mediate the Aspergillus branching response; one of the mutants is locked in a hypo-branching phenotype, while the other two mutants display a hyper-branching phenotype. Our work reveals an endogenous signal that triggers crucial developmental processes in filamentous fungi, and opens new avenues for research on the morphogenesis of filamentous fungi.

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Section: Methodsmentioning

confidence: 99%

Fungal oxylipins direct programmed developmental switches in filamentous fungi

et al. 2020

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“…Fraczek, M. G., Zhao, C., Dineen, L., Lebedinec, R., Bowyer, P., Bromley, M., & Delneri, D. (2019). Fast and reliable PCR amplification from Aspergillus fumigatus spore suspension without traditional DNA extraction.…”

Section: How To Cite This Articlementioning

confidence: 99%

“…3A and 3B). Primers hphsqR (5 -CACCGGTCAACCATGATCTG-3 ) and hphsqF (5 -CCCAGCACTCGTCCGAGGG C-3 ) target the marker gene hygromycin B phosphotransferase (hph), which was used to replace the gene of interest [see Current Protocols article Zhao et al (2019) for detailed protocol]. Primers P1 and P4 are specific for the upstream and downstream flank sequences of the deleted gene, respectively.…”

Section: Of 10mentioning

confidence: 99%

Fast and Reliable PCR Amplification from Aspergillus fumigatus Spore Suspension Without Traditional DNA Extraction

et al. 2019

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Aspergillus fumigatus is an opportunistic human pathogenic mold. DNA extraction from this fungus is usually performed by mechanical perturbation of cells, as it possesses a rigid and complex cell wall. While this is not problematic for single isolates, it can be time consuming for large numbers of strains if using traditional DNA extraction procedures. Therefore, in this article we describe a fast and efficient thermal‐shock method to release DNA from spores of A. fumigatus and other filamentous fungi without the need for complex extraction methods. This is especially important for high‐throughput PCR analyses of mutants in 96‐ or 384‐well formats in a very short period of time without any concern about sample cross‐contamination. This method is currently being used to validate the protein‐coding gene and non‐coding RNA knockout libraries in A. fumigatus generated in our laboratory, and could be used in the future for diagnostics purposes. © 2019 The Authors.

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“…Strains and media. The Aspergillus fumigatus transcription factor deletant library, derived from wild-type strain MFIG001, was constructed by homologous recombination using gene replacement cassettes and transformation methodologies described previously (30,41). Studies in Aspergillus niger were performed in the A. niger N402 background (referred to as the A. niger WT throughout) and an A. niger ΔcdcA mutant strain (14).…”

Section: Methodsmentioning

confidence: 99%

Weak Acid Resistance A (WarA), a Novel Transcription Factor Required for Regulation of Weak-Acid Resistance and Spore-Spore Heterogeneity in Aspergillus niger

Geoghegan

Stratford

Bromley

et al. 2020

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Propionic, sorbic, and benzoic acids are organic weak acids that are widely used as food preservatives, where they play a critical role in preventing microbial growth. In this study, we uncovered new mechanisms of weak-acid resistance in molds. By screening a library of 401 transcription factor deletion strains in Aspergillus fumigatus for sorbic acid hypersensitivity, a previously uncharacterized transcription factor was identified and named weak acid resistance A (WarA). The orthologous gene in the spoilage mold Aspergillus niger was identified and deleted. WarA was required for resistance to a range of weak acids, including sorbic, propionic, and benzoic acids. A transcriptomic analysis was performed to characterize genes regulated by WarA during sorbic acid treatment in A. niger. Several genes were significantly upregulated in the wild type compared with a ΔwarA mutant, including genes encoding putative weak-acid detoxification enzymes and transporter proteins. Among these was An14g03570, a putative ABC-type transporter which we found to be required for weak-acid resistance in A. niger. We also show that An14g03570 is a functional homologue of the Saccharomyces cerevisiae protein Pdr12p and we therefore name it PdrA. Last, resistance to sorbic acid was found to be highly heterogeneous within genetically uniform populations of ungerminated A. niger conidia, and we demonstrate that pdrA is a determinant of this heteroresistance. This study has identified novel mechanisms of weak-acid resistance in A. niger which could help inform and improve future food spoilage prevention strategies. IMPORTANCE Weak acids are widely used as food preservatives, as they are very effective at preventing the growth of most species of bacteria and fungi. However, some species of molds can survive and grow in the concentrations of weak acid employed in food and drink products, thereby causing spoilage with resultant risks for food security and health. Current knowledge of weak-acid resistance mechanisms in these fungi is limited, especially in comparison to that in yeasts. We characterized gene functions in the spoilage mold species Aspergillus niger which are important for survival and growth in the presence of weak-acid preservatives. Such identification of weak-acid resistance mechanisms in spoilage molds will help in the design of new strategies to reduce food spoilage in the future.

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High‐Throughput Gene Replacement in Aspergillus fumigatus

Cited by 45 publications

References 18 publications

Fungal oxylipins direct programmed developmental switches in filamentous fungi

Fungal oxylipins direct programmed developmental switches in filamentous fungi

Fast and Reliable PCR Amplification from Aspergillus fumigatus Spore Suspension Without Traditional DNA Extraction

Weak Acid Resistance A (WarA), a Novel Transcription Factor Required for Regulation of Weak-Acid Resistance and Spore-Spore Heterogeneity in Aspergillus niger

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