title: High-throughput FACS-based mutant screen identifies a gain-of-function allele of the Fusarium graminearum adenylyl cyclase causing deoxynivalenol over-production.
abstract: Fusarium head blight and crown rot, caused by the fungal plant pathogen Fusarium graminearum, impose a major threat to global wheat production. During the infection, plants are contaminated with mycotoxins such as deoxynivalenol (DON), which can be toxic for humans and animals. In addition, DON is a major virulence factor during wheat infection. However, it is not fully understood how DON production is regulated in F. graminearum. In order to identify regulators of DON production, a high-throughput mutant screen using Fluorescence Activated Cell Sorting (FACS) of a mutagenised TRI5-GFP reporter strain was established and a mutant over-producing DON under repressive conditions identified. A gain-of-function mutation in the F. graminearum adenylyl cyclase (FAC1), which is a known positive regulator of DON production, was identified as the cause of this phenotype through genome sequencing and segregation analysis. Our results show that the high-throughput mutant screening procedure developed here can be applied for identification of fungal proteins involved in diverse processes.
authors.affiliation: CSIRO Agriculture, Queensland Bioscience Precinct, 306 Carmody Road, St Lucia, Brisbane, Queensland 4067, Australia; School of Agriculture & Food Sciences, University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia. Electronic address: Ailisa.Blum@csiro.au.
authors.forename: Ailisa
authors.initials: A
authors.lastname: Blum
authors.affiliation: CSIRO Agriculture, Queensland Bioscience Precinct, 306 Carmody Road, St Lucia, Brisbane, Queensland 4067, Australia.
authors.forename: Aurélie H
authors.initials: AH
authors.lastname: Benfield
authors.affiliation: CSIRO Agriculture, Queensland Bioscience Precinct, 306 Carmody Road, St Lucia, Brisbane, Queensland 4067, Australia.
authors.forename: Jiri
authors.initials: J
authors.lastname: Stiller
authors.affiliation: CSIRO Agriculture, Queensland Bioscience Precinct, 306 Carmody Road, St Lucia, Brisbane, Queensland 4067, Australia; Queensland Alliance for Agriculture & Food Innovation (QAAFI), University of Queensland, St Lucia, Brisbane, Queensland 4067, Australia.
authors.forename: Kemal
authors.initials: K
authors.lastname: Kazan
authors.affiliation: School of Agriculture & Food Sciences, University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia; School of Plant Biology, University of Western Australia, Crawley, Western Australia 6009, Australia.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Batley
authors.affiliation: CSIRO Agriculture, Queensland Bioscience Precinct, 306 Carmody Road, St Lucia, Brisbane, Queensland 4067, Australia.
authors.forename: Donald M
authors.initials: DM
authors.lastname: Gardiner
mesh_terms: D000249:Adenosine Monophosphate / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000235:genetics* / Q000378:metabolism*; D000483:Alleles; D004271:DNA, Fungal; D005434:Flow Cytometry / Q000379:methods; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005670:Fusarium / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods; D009154:Mutation; D009183:Mycotoxins / Q000378:metabolism; D010641:Phenotype; D010935:Plant Diseases / Q000382:microbiology; D014255:Trichothecenes / Q000096:biosynthesis* / Q000378:metabolism; D014908:Triticum / Q000382:microbiology
chemical_list: D004271:DNA, Fungal; D005656:Fungal Proteins; D009183:Mycotoxins; D014255:Trichothecenes; D000249:Adenosine Monophosphate; D000262:Adenylyl Cyclases; C007262:deoxynivalenol
keywords: DON; Flow cytometry; Fluorescence activated cell sorting; Gibberella zeae; Trichothecene; cAMP