High-throughput FACS-based mutant screen identifies a gain-of-function allele of the Fusarium graminearum adenylyl cyclase causing deoxynivalenol over-production

Blum, Ailisa; Benfield, Aurélie H.; Stiller, Jiri; Kazan, Kemal; Batley, Jacqueline; Gardiner, Donald M.

doi:10.1016/j.fgb.2016.02.005

Cited by 21 publications

(21 citation statements)

References 67 publications

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“…Nevertheless, in F. pseudograminearum FCK1-GFP is expressed in these ovoid cells and is localised in the cytosol, similar to TRI5-GFP in F. graminearum (Blum et al, 2016;Boenisch et al, 2017).…”

Section: Discussionmentioning

confidence: 87%

“…Similar to the DON biosynthesis enzyme TRI5 in F. graminearum (Blum et al, 2016;Boenisch et al, 2017), the F. pseudograminearum FCK1-GFP is also localized in the cytosol (Figure 4.3). Under the fusatin biosynthesis inducing conditions F. pseudograminearum also developed ovoid cells (Figure 4.3, second row), which are similar to those previously observed in F. graminearum during DON biosynthesis inducing conditions (Blum et al, 2016;Menke et al, 2013). …”

Section: The Fusatin Biosynthesis Enzyme Fck1 Is Localized In the Cytmentioning

confidence: 95%

“…Further, adenylyl cyclase mutants are not only altered in DON production, but also in cellular development, as the ∆fac1 knockout mutant is unable to produce infection structures (Bormann et al, 2014) and the gain of function mutant develops ovoid and bulbous cells during toxin production repressing conditions (Blum et al, 2016). It will be discussed below, how cAMP signalling could regulate cellular development.…”

Section: F Graminearummentioning

confidence: 99%

“…TRI1 and TRI4 are both cytochrome P450 monooxygenases which catalyse the eleventh and twelfth and the second through fifth DON biosynthesis steps respectively (reviewed in Desjardins, 2006). However, TRI5 which catalyses the first DON biosynthesis step, is localized in the cytosol (Blum et al, 2016;Boenisch et al, 2017). This raises the question how the toxin biosynthesis is coordinated between the cytosol and the smooth ER proliferations.…”

Section: Introductionmentioning

confidence: 99%

“…As a similar biosynthesis regulation through specific nitrogen sources and pH had been observed before during DON production in F. graminearum, the production levels of fusatin and DON under these different axenic culture conditions were quantified in both F. pseudograminearum and F. graminearum. FgTRI5-GFP also carries a translational C-terminal GFP fusion in the endogenous FgTRI5 locus in the CS3005 F. graminearum wild type background (Blum et al, 2016). The wheat cultivar Kennedy was used for the wheat seedling infections.…”

Section: Introductionmentioning

confidence: 99%

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Regulation and cell biology of secondary metabolite production in Fusarium graminearum and Fusarium pseudograminearum

Blum¹

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1 AbstractAscomycete fungi have the potential to produce a vast array of secondary metabolites, which are metabolites not required for normal growth and development. In any one species there is typically the potential for production of upward of fifty of these compounds. Many fungal secondary metabolites are used as medicines (e.g. penicillin or cyclosporine), or are of importance due to their toxicity in humans or animals (e.g. aflatoxins), or because they are virulence factors during crop plant infection (e.g. deoxynivalenol). Despite the vast potential encoded in ascomycete genomes, the vast majority of fungal secondary metabolites are yet to be discovered. This is in part due to our lack of understanding of how the production of these compounds is regulated. Moreover, even for the compounds we do know, we are just beginning to understand the complex interaction of external stimuli, signalling pathways, cellular and developmental biology that leads to their production. In this thesis, biological insights into these aspects of two fungal secondary metabolites, deoxynivalenol (DON) and fusatin are provided.In chapter II a high-throughput fluorescence activated cell sorting (FACS) based mutant screen was developed and the regulation of DON production in Fusarium graminearum was analysed. DON is an economically very important mycotoxin, as it is the most common contaminant of wheat, barley and corn worldwide. It is produced by plant pathogenic filamentous fungi of the Fusarium family, which can cause Fusarium head blight as well as Fusarium crown rot. During plant infection, DON is an important virulence factor and it can be toxic for humans and animals. The regulation of DON production is not yet fully understood. To identify regulators, a mutant screen was developed using FACS to allow high throughput screening, coupled with whole genome sequencing to identify mutations.Segregation analyses were performed to determine the mutation causing the phenotype.The mutant screen identified an adenylyl cyclase allele, which resulted in production of DON under normally repressive conditions. In addition, the mutant developed cellular structures associated with toxin production under repressive conditions. The levels of the fundamental second messenger cAMP, which adenylyl cyclases produce, were increased in the identified mutant, suggesting it might be a gain-of-function adenylyl cyclase mutant. This mutant screening methodology can be adapted to identify regulators of different secondary metabolites as well as to identify mutants overproducing yet unknown secondary metabolites. Abstract 2In chapter III the cell biology of DON production in Fusarium graminearum was elucidated with quantitative super resolution microscopy. During the last five years the first insights into the cell biology of DON production were gained, indicating complex cell biological mechanisms. During toxin production, F. graminearum develops endoplasmic reticulum proliferations, called toxisomes, at which two DON biosynthesis enzymes, TRI1 a...

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Section: Discussionmentioning

confidence: 87%

Section: The Fusatin Biosynthesis Enzyme Fck1 Is Localized In the Cytmentioning

confidence: 95%

Section: F Graminearummentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Regulation and cell biology of secondary metabolite production in Fusarium graminearum and Fusarium pseudograminearum

Blum¹

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Enhanced avermectin production by Streptomyces avermitilis ATCC 31267 using high-throughput screening aided by fluorescence-activated cell sorting

Cao

Luo

Zeng

et al. 2017

Appl Microbiol Biotechnol

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Avermectins, produced by Streptomyces avermitilis, are important antiparasitic agents. The use of traditional microbial breeding methods for this organism has been limited by the low-throughput shake flask-based screening process. The unique growth cycle of actinomycetes makes the establishment of a reliable high-throughput screening (HTS) process difficult. To enhance the efficiency of screening strains with high yields of avermectin, a HTS process aided by fluorescence-activated cell sorting (FACS) was established. Four different spore solutions were investigated for maintaining a relatively high viability of spores. Propidium iodide (PI) and fluorescein diacetate (FDA) were used to discriminate between dead and live spores using the FACS system. Spores stained with 7-μg/mL PI and 15-μg/mL FDA at 4 °C in the dark for 30 min resulted in optimum sorting. Spores were treated by atmospheric and room temperature plasma (ARTP). Single live spores were sorted and sprayed into 96-well microtiter plates containing 50 μL of solid agar culture medium. Solid-liquid combinatorial microculture was used for high-throughput avermectin culture. A high-titer avermectin producer (G9) was obtained from 5760 mutants after mutagenesis and HTS. Compared with the original strain, the titer was improved by 18.9% on flask culture and 20.6% on fermenter, respectively. The HTS process established in this study could easily be transferred to other similar target products produced by actinomycetes.

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Development of a flow cytometry-based plating-free system for strain engineering in industrial fungi

Yin

Liu

et al. 2021

Appl Microbiol Biotechnol

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High-throughput FACS-based mutant screen identifies a gain-of-function allele of the Fusarium graminearum adenylyl cyclase causing deoxynivalenol over-production

Cited by 21 publications

References 67 publications

Regulation and cell biology of secondary metabolite production in Fusarium graminearum and Fusarium pseudograminearum

Regulation and cell biology of secondary metabolite production in Fusarium graminearum and Fusarium pseudograminearum

Enhanced avermectin production by Streptomyces avermitilis ATCC 31267 using high-throughput screening aided by fluorescence-activated cell sorting

Development of a flow cytometry-based plating-free system for strain engineering in industrial fungi

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