Utilizing next generation sequencing to revitalize a forward genetic screen for mutants deficient in the production of sterigmatocystin in Aspergillus nidulans

B. T. Pfannenstiela, K. J. Affeldtb, J. Bokb, R. A. E. Butchkoc, T. Choerab, G. J. Fischera, B. P. Knoxb, F. Y. Limb, X. Luod, A. A. Soukupa, J. E. Sprakerd, K. Throckmortona, P. Wiemannb, N. P. Kellerb,e, and J. M. Palmerf

Author address: 

aDepartment of Genetics, University of Wisconsin-Madison, Madison, WI, 53706, USA. bDepartment of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI, 53706, USA. cDepartment of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77843, USA. dDepartment of Plant Pathology, University of Wisconsin-Madison, Wisconsin 53706. eDepartment of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA. fCenter for Forest Mycology Research, Northern Research Station, U.S. Forest Service, Madison, WI 53706, USA.

Abstract: 

The study of aflatoxin regulation in Aspergillus spp. has warranted the attention of many researchers due to its carcinogenic properties and frequency as a food and feed contaminant. Significant progress on the regulation of the AF cluster has been conducted in the model organism Aspergillus nidulans by assessing regulation of the sterigmatocystin (ST) gene cluster as ST is the penultimate precursor of AF. A screen using a chemical mutagen identified 23 loci involved in regulating ST production. Only six of these loci were characterized from this screen using classical mapping (mcsA) and complementation with a cosmid library (laeA). Recently the remaining mutants were backcrossed and sequenced using an Ion Torrent PGMTM. Each mutant contained one or more SNPs in predicted genes. Deletion of these genes resulted in identification of mutant alleles responsible for the loss of ST production in 12 out of the 17 remaining mutants. Three of the causative mutations were in uncharacterized genes unknown to be involved with ST production, of which two appear to regulate the ST cluster via transcriptional regulation of its cluster specific transcription factor, aflR. Based on protein domains and homologs, the remaining uncharacterized gene has a predicted role in the plasma membrane fusion. Nine mutations were in genes already known to affect ST synthesis (laeA, mcsA, fluG, stcA).

2017

abstract No: 

62

Full conference title: 

The Fourteenth International Aspergillus Meeting, Asilomar Conference Center, Pacific Grove, CA, USA
    • Asperfest 14 (2017)