Revitalization of a Forward Genetic Screen Identifies Three New Regulators of Fungal Secondary Metabolism in the Genus
Aspergillus
Authors: Pfannenstiel Brandon T. , Zhao Xixi , Wortman Jennifer , Wiemann Philipp , Throckmorton Kurt , Spraker Joseph E. , Soukup Alexandra A. , Luo Xingyu , Lindner Daniel L. , Lim Fang Yun , Knox Benjamin P. , Haas Brian , Fischer Gregory J. , Choera Tsokyi , Butchko Robert A. E. , Bok Jin-Woo , Affeldt Katharyn J. , Keller Nancy P. , Palmer Jonathan M. 
Published: September 2017

ABSTRACT

The 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.