Revitalization of a Forward Genetic Screen Identifies Three New Regulators of Fungal Secondary Metabolism in the Genus Aspergillus

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 wholegenome 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

New Insight Into Pathogenicity and Secondary Metabolism of the Plant Pathogen Penicillium expansum Through Deletion of the Epigenetic Reader SntB

“…Production of both mycotoxins was recovered by the complement strain, although not to the level of the control strain, which was in agreement with gene expression analysis (Figures 2A-D). These results were not totally unexpected considering that previous publications have reported similar outcomes, where the complement strains partially restored the parental phenotype (Carvalho et al, 2010;Kwon et al, 2011;Pfannenstiel et al, 2017).…”

“…Most studies investigated the role of histone modifying enzymes, such as methylases or histone deacetylases in modulating secondary metabolite synthesis in fungi. However, more recently an additional histone modifying protein, the epigenetic reader SntB, was identified in Aspergillus species to regulate not only fungal development and secondary metabolism (Pfannenstiel et al, 2017) but also virulence in the aflatoxigenic fungus A. flavus (Pfannenstiel et al, 2018). SntB orthologs, called Snt2, had previously been described in the plant pathogens Fusarium oxysporum (Denisov et al, 2011) and Magnaporthe oryzae (He et al, 2018).…”

Global population genomics of the forest pathogen Dothistroma septosporum reveal chromosome duplications in high dothistromin‐producing strains

“…Some genes with potential roles in boosting dothistromin production are present on chromosome 11, although these duplications were shared with two other strains: USA12 (lowest dothistromin producer) and SAF4 (high producer). Amongst these genes, Ds75171 is an orthologue of A. nidulans AN5169 (Boi1), a phospholipid binding protein gene recently implicated in regulation of secondary metabolism (Pfannenstiel et al ., ). Another gene present on chromosome 11 that could facilitate high levels of dothistromin production is a putative major facilitator superfamily (MFS) transporter gene Ds75255.…”

A new regulator RsdA mediating fungal secondary metabolism has a detrimental impact on asexual development in Pestalotiopsis fici

“…S5), which indicated that it was a new functional protein. The protein in P. fici consisted of 730 amino acids encoded by 2779 nucleotides, with three putative introns and a possible Aspergillus nidulans homologue of this protein (AN4699) has just been reported, but no clear function was described (Pfannenstiel et al, ). Collectively, these results show that RsdA is a new conserved regulator in fungi.…”