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dc.contributor.authorPfannenstiel, Brandon T.
dc.contributor.authorZhao, Xixi
dc.contributor.authorWortman, Jennifer
dc.contributor.authorWiemann, Philipp
dc.contributor.authorThrockmorton, Kurt
dc.contributor.authorSpraker, Joseph E.
dc.contributor.authorSoukup, Alexandra A.
dc.contributor.authorLuo, Xingyu
dc.contributor.authorLindner, Daniel L.
dc.contributor.authorLim, Fang Yun
dc.contributor.authorKnox, Benjamin P.
dc.contributor.authorHaas, Brian
dc.contributor.authorFischer, Gregory J.
dc.contributor.authorChoera, Tsokyi
dc.contributor.authorButchko, Robert A. E.
dc.contributor.authorBok, Jin-Woo
dc.contributor.authorAffeldt, Katharyn J.
dc.contributor.authorKeller, Nancy P.
dc.contributor.authorPalmer, Jonathan M.
dc.date.accessioned2018-01-31T16:30:59Z
dc.date.available2018-01-31T16:30:59Z
dc.date.issued2017-09-05
dc.identifier.citationRevitalization of a Forward Genetic Screen Identifies Three New Regulators of Fungal Secondary Metabolism in the Genus Aspergillus 2017, 8 (5):e01246-17 mBioen
dc.identifier.issn2150-7511
dc.identifier.doi10.1128/mBio.01246-17
dc.identifier.urihttp://hdl.handle.net/10150/626452
dc.description.abstractThe study of aflatoxin in Aspergillus spp. has garnered the attention of many researchers due to aflatoxin's carcinogenic properties and frequency as a food and feed contaminant. Significant progress has been made by utilizing the model organism Aspergillus nidulans to characterize the regulation of sterigmatocystin (ST), the penultimate precursor of aflatoxin. A previous forward genetic screen identified 23 A. nidulans mutants involved in regulating ST production. Six mutants were characterized from this screen using classical mapping (five mutations in mcsA) and complementation with a cosmid library (one mutation in laeA). The remaining mutants were backcrossed and sequenced using Illumina and Ion Torrent sequencing platforms. All but one mutant contained one or more sequence variants in predicted open reading frames. Deletion of these genes resulted in identification of mutant alleles responsible for the loss of ST production in 12 of the 17 remaining mutants. Eight of these mutations were in genes already known to affect ST synthesis (laeA, mcsA, fluG, and stcA), while the remaining four mutations (in laeB, sntB, and hamI) were in previously uncharacterized genes not known to be involved in ST production. Deletion of laeB, sntB, and hamI in A. flavus results in loss of aflatoxin production, confirming that these regulators are conserved in the aflatoxigenic aspergilli. This report highlights the multifaceted regulatory mechanisms governing secondary metabolism in Aspergillus. Additionally, these data contribute to the increasing number of studies showing that forward genetic screens of fungi coupled with whole-genome resequencing is a robust and cost-effective technique. IMPORTANCE In a postgenomic world, reverse genetic approaches have displaced their forward genetic counterparts. The techniques used in forward genetics to identify loci of interest were typically very cumbersome and time-consuming, relying on Mendelian traits in model organisms. The current work was pursued not only to identify alleles involved in regulation of secondary metabolism but also to demonstrate a return to forward genetics to track phenotypes and to discover genetic pathways that could not be predicted through a reverse genetics approach. While identification of mutant alleles from whole-genome sequencing has been done before, here we illustrate the possibility of coupling this strategy with a genetic screen to identify multiple alleles of interest. Sequencing of classically derived mutants revealed several uncharacterized genes, which represent novel pathways to regulate and control the biosynthesis of sterigmatocystin and of aflatoxin, a societally and medically important mycotoxin.
dc.description.sponsorshipNational Institute of General Medical Sciences [PO1-GM084077]; UW-Madison Food Research Institute; USDA Hatch Formula Fund [WIS01710]; Predoctoral Training Program in Genetics - National Institutes of Health [5 T32 GM007133-40]; National Institutes of Health [1R01GM112739-01]; NSF [DGE-1256259]; U.S. Forest Service, Northern Research Stationen
dc.language.isoenen
dc.publisherAMER SOC MICROBIOLOGYen
dc.relation.urlhttp://mbio.asm.org/lookup/doi/10.1128/mBio.01246-17en
dc.rights© The Author(s). ASM publishes mBio articles under the Creative Commons Attribution license. Starting in 2016, articles are covered under a Creative Commons Attribution 4.0 International license.en
dc.subjectAspergillus nidulansen
dc.subjectforward geneticsen
dc.subjectwhole-genome sequencingen
dc.subjectsecondary metabolismen
dc.titleRevitalization of a Forward Genetic Screen Identifies Three New Regulators of Fungal Secondary Metabolism in the Genus Aspergillusen
dc.typeArticleen
dc.contributor.departmentUniv Arizona, Sch Plant Scien
dc.identifier.journalmBioen
dc.description.collectioninformationThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at repository@u.library.arizona.edu.en
dc.eprint.versionFinal published versionen
refterms.dateFOA2018-08-16T21:49:49Z
html.description.abstractThe study of aflatoxin in Aspergillus spp. has garnered the attention of many researchers due to aflatoxin's carcinogenic properties and frequency as a food and feed contaminant. Significant progress has been made by utilizing the model organism Aspergillus nidulans to characterize the regulation of sterigmatocystin (ST), the penultimate precursor of aflatoxin. A previous forward genetic screen identified 23 A. nidulans mutants involved in regulating ST production. Six mutants were characterized from this screen using classical mapping (five mutations in mcsA) and complementation with a cosmid library (one mutation in laeA). The remaining mutants were backcrossed and sequenced using Illumina and Ion Torrent sequencing platforms. All but one mutant contained one or more sequence variants in predicted open reading frames. Deletion of these genes resulted in identification of mutant alleles responsible for the loss of ST production in 12 of the 17 remaining mutants. Eight of these mutations were in genes already known to affect ST synthesis (laeA, mcsA, fluG, and stcA), while the remaining four mutations (in laeB, sntB, and hamI) were in previously uncharacterized genes not known to be involved in ST production. Deletion of laeB, sntB, and hamI in A. flavus results in loss of aflatoxin production, confirming that these regulators are conserved in the aflatoxigenic aspergilli. This report highlights the multifaceted regulatory mechanisms governing secondary metabolism in Aspergillus. Additionally, these data contribute to the increasing number of studies showing that forward genetic screens of fungi coupled with whole-genome resequencing is a robust and cost-effective technique. IMPORTANCE In a postgenomic world, reverse genetic approaches have displaced their forward genetic counterparts. The techniques used in forward genetics to identify loci of interest were typically very cumbersome and time-consuming, relying on Mendelian traits in model organisms. The current work was pursued not only to identify alleles involved in regulation of secondary metabolism but also to demonstrate a return to forward genetics to track phenotypes and to discover genetic pathways that could not be predicted through a reverse genetics approach. While identification of mutant alleles from whole-genome sequencing has been done before, here we illustrate the possibility of coupling this strategy with a genetic screen to identify multiple alleles of interest. Sequencing of classically derived mutants revealed several uncharacterized genes, which represent novel pathways to regulate and control the biosynthesis of sterigmatocystin and of aflatoxin, a societally and medically important mycotoxin.


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