Production of cyclopiazonic acid,aflatrem, and aflatoxin by <Emphasis Type="Italic">Aspergillus flavus</Emphasis> is regulated by <Emphasis Type="Italic">veA</Emphasis>, a gene necessary for sclerotial formation

The plant pathogenic fungus Aspergillus flavus produces several types of mycotoxins. The most well known are the carcinogenic compounds called aflatoxins. In addition, A. flavus produces cyclopiazonic acid and aflatrem mycotoxins, contributing to the toxicity of A. flavus infected crops. Cyclopiazonic acid is a specific inhibitor of calcium-dependent ATPase in the sarcoplasmic reticulum that results in altered cellular Ca⁺⁺ levels. Aflatrem is a potent tremorgenic mycotoxin known to lead to neurological disorders. Previously we showed that a gene called veA controls aflatoxin and sclerotial production in A. parasiticus. In this study in A. flavus, we show that the veA homolog in A. flavus not only is necessary for the production of aflatoxins B1 and B2 and sclerotia, but also regulates the synthesis of the mycotoxins cyclopiazonic acid and aflatrem. The A. flavus ΔveA mutant was completely blocked in the production of aflatrem and showed greater than twofold decrease in cyclopiazonic acid production. The genes involved in the synthesis of cyclopiazonic acid are unknown; however, the aflatrem gene cluster has been characterized. Northern hybridization analysis showed that veA is required for expression of the A. flavus aflatrem genes atmC, atmG, and atmM. This is the first report of a regulatory gene governing the production of cyclopiazonic acid and aflatrem mycotoxins.     

Origin and distribution of epipolythiodioxopiperazine (ETP) gene clusters in filamentous ascomycetes

Background  

Genes responsible for biosynthesis of fungal secondary metabolites are usually tightly clustered in the genome and co-regulated with metabolite production. Epipolythiodioxopiperazines (ETPs) are a class of secondary metabolite toxins produced by disparate ascomycete fungi and implicated in several animal and plant diseases. Gene clusters responsible for their production have previously been defined in only two fungi. Fungal genome sequence data have been surveyed for the presence of putative ETP clusters and cluster data have been generated from several fungal taxa where genome sequences are not available. Phylogenetic analysis of cluster genes has been used to investigate the assembly and heredity of these gene clusters.     

VeA and MvlA repression of the cryptic orsellinic acid gene cluster in Aspergillus nidulans involves histone 3 acetylation

A perplexing aspect of fungal secondary metabolite gene clusters is that most clusters remain ‘silent’ under common laboratory growth conditions where activation is obtained through gene manipulation or encounters with environmental signals. Few proteins have been found involved in repression of silent clusters. Through multicopy suppressor mutagenesis, we have identified a novel cluster suppressor in Aspergillus nidulans, MvlA (m odulator of v eA l oss). Genetic assessment of MvlA mutants revealed the role of both itself and VeA (but not the VeA partner LaeA) in the suppression of the cryptic ors gene cluster producing orsellinic acid and its F9775 derivatives. Loss of veA upregulates F9775A and F9775B production and this increase is reduced 4–5‐fold when an overexpression mvlA (OE:mvlA) allele is introduced into the ΔveA background. Previous studies have implicated a positive role for GcnE (H3K9 acetyltransferase of the SAGA/ADA complex) in ors cluster expression and here we find expression of gcnE is upregulated in ΔveA and suppressed by OE:mvlA in the ΔveA background. H3K9 acetylation levels of ors cluster genes correlated with gcnE expression and F9775 production in ΔveA and OE:mvlAΔveA strains. Finally, deletion of gcnE in the ΔveA background abolishes ors cluster activation and F9775 production. Together, this work supports a role for VeA and MvlA in modifying SAGA/ADA complex activity.     

veA Is Required for Toxin and Sclerotial Production in Aspergillus parasiticus

It was long been noted that secondary metabolism is associated with fungal development. In Aspergillus nidulans, conidiation and mycotoxin production are linked by a G protein signaling pathway. Also in A. nidulans, cleistothecial development and mycotoxin production are controlled by a gene called veA. Here we report the characterization of a veA ortholog in the aflatoxin-producing fungus A. parasiticus. Cleistothecia are not produced by Aspergillus parasiticus; instead, this fungus produces spherical structures called sclerotia that allow for survival under adverse conditions. Deletion of veA from A. parasiticus resulted in the blockage of sclerotial formation as well as a blockage in the production of aflatoxin intermediates. Our results indicate that A. parasiticus veA is required for the expression of aflR and aflJ, which regulate the activation of the aflatoxin gene cluster. In addition to these findings, we observed that deletion of veA reduced conidiation both on the culture medium and on peanut seed. The fact that veA is necessary for conidiation, production of resistant structures, and aflatoxin biosynthesis makes veA a good candidate gene to control aflatoxin biosynthesis or fungal development and in this way to greatly decrease its devastating impact on health and the economy.     

Genetic regulation of aflatoxin biosynthesis: From gene to genome

Aflatoxins are notorious toxic secondary metabolites known for their impacts on human and animal health, and their effects on the marketability of key grain and nut crops. Understanding aflatoxin biosynthesis is the focus of a large and diverse research community. Concerted efforts by this community have led not only to a well-characterized biosynthetic pathway, but also to the discovery of novel regulatory mechanisms. Common to secondary metabolism is the clustering of biosynthetic genes and their regulation by pathway specific as well as global regulators. Recent data show that arrangement of secondary metabolite genes in clusters may allow for an important global regulation of secondary metabolism based on physical location along the chromosome. Available genomic and proteomic tools are now allowing us to examine aflatoxin biosynthesis more broadly and to put its regulation in context with fungal development and fungal ecology. This review covers our current understanding of the biosynthesis and regulation of aflatoxin and highlights new and emerging information garnered from structural and functional genomics. The focus of this review will be on studies in Aspergillus flavus and Aspergillus parasiticus, the two agronomically important species that produce aflatoxin. Also covered will be the important contributions gained by studies on production of the aflatoxin precursor sterigmatocystin in Aspergillus nidulans.     

Clustered genes involved in cyclopiazonic acid production are next to the aflatoxin biosynthesis gene cluster in Aspergillus flavus

Cyclopiazonic acid (CPA), an indole-tetramic acid mycotoxin, is produced by many species of Aspergillus and Penicillium. In addition to CPA Aspergillus flavus produces polyketide-derived carcinogenic aflatoxins. Aflatoxin biosynthesis genes form a gene cluster in a subtelomeric region. Isolates of A. flavus lacking aflatoxin production due to the loss of the entire aflatoxin gene cluster and portions of the subtelomeric region are often unable to produce CPA, which suggests a physical link of genes involved in CPA biosynthesis to the aflatoxin gene cluster. Examining the subtelomeric region in A. flavus isolates of different chemotypes revealed a region possibly associated with CPA production. Disruption of three of the four genes present in this region predicted to encode a monoamine oxidase, a dimethylallyl tryptophan synthase, and a hybrid polyketide non-ribosomal peptide synthase abolished CPA production in an aflatoxigenic A. flavus strain. Therefore, some of the CPA biosynthesis genes are organized in a mini-gene cluster that is next to the aflatoxin gene cluster in A. flavus.     

Heterochromatin influences the secondary metabolite profile in the plant pathogen Fusarium graminearum

Chromatin modifications and heterochromatic marks have been shown to be involved in the regulation of secondary metabolism gene clusters in the fungal model system Aspergillus nidulans. We examine here the role of HEP1, the heterochromatin protein homolog of Fusarium graminearum, for the production of secondary metabolites. Deletion of Hep1 in a PH-1 background strongly influences expression of genes required for the production of aurofusarin and the main tricothecene metabolite DON. In the Hep1 deletion strains AUR genes are highly up-regulated and aurofusarin production is greatly enhanced suggesting a repressive role for heterochromatin on gene expression of this cluster. Unexpectedly, gene expression and metabolites are lower for the trichothecene cluster suggesting a positive function of Hep1 for DON biosynthesis. However, analysis of histone modifications in chromatin of AUR and DON gene promoters reveals that in both gene clusters the H3K9me3 heterochromatic mark is strongly reduced in the Hep1 deletion strain. This, and the finding that a DON-cluster flanking gene is up-regulated, suggests that the DON biosynthetic cluster is repressed by HEP1 directly and indirectly. Results from this study point to a conserved mode of secondary metabolite (SM) biosynthesis regulation in fungi by chromatin modifications and the formation of facultative heterochromatin.     

黄曲霉功能基因组学研究

黄曲霉（Aspergillus flavus）是一种常见的腐生真菌和条件致病菌,其次生代谢产物黄曲霉毒素（Aflatoxin,AFT）具有高度的致癌性和致畸性,严重危及人类和动物健康。近年来,功能基因组学研究发展迅速,在真菌生长发育、挖掘真菌次级代谢产物以及研究包括黄曲霉毒素在内的真菌毒素等方面得到了广泛的应用。功能基因组学在研究黄曲霉与宿主之间的相互作用以及黄曲霉与其他曲霉之间的相互作用方面具有巨大的潜力。然而,黄曲霉功能基因组学受到细胞壁难以破除、耐药性高、筛选标记少、缺陷型菌株构建费力耗时等因素的影响而发展缓慢。概述了黄曲霉的选择标记、遗传转化方法和黄曲霉毒素以及环匹阿尼酸（cyclopiazonic acid, CPA）生物合成的研究进展,并讨论了在提高黄曲霉基因操作效率方面的潜在策略。例如,构建缺乏非同源末端连接（NHEJ）途径的菌株、Cre-loxP重组系统、CRISPR-Cas9等方法,为深入开展黄曲霉遗传学研究提供参考。