Transcriptome Analysis of Aspergillus flavus Reveals veA -Dependent Regulation of Secondary Metabolite Gene Clusters, Including the Novel Aflavarin Cluster

Abstract

eThe global regulatory veA gene governs development and secondary metabolism in numerous fungal species, including Aspergillus flavus. This is especially relevant since A. flavus infects crops of agricultural importance worldwide, contaminating them with potent mycotoxins. The most well-known are aflatoxins, which are cytotoxic and carcinogenic polyketide compounds. The production of aflatoxins and the expression of genes implicated in the production of these mycotoxins are veA dependent. The genes responsible for the synthesis of aflatoxins are clustered, a signature common for genes involved in fungal secondary metabolism. Studies of the A. flavus genome revealed many gene clusters possibly connected to the synthesis of secondary metabolites. Many of these metabolites are still unknown, or the association between a known metabolite and a particular gene cluster has not yet been established. In the present transcriptome study, we show that veA is necessary for the expression of a large number of genes. Twenty-eight out of the predicted 56 secondary metabolite gene clusters include at least one gene that is differentially expressed depending on presence or absence of veA. One of the clusters under the influence of veA is cluster 39. The absence of veA results in a downregulation of the five genes found within this cluster. Interestingly, our results indicate that the cluster is expressed mainly in sclerotia. Chemical analysis of sclerotial extracts revealed that cluster 39 is responsible for the production of aflavarin.A spergillus flavus is a saprophytic filamentous fungus that is also able to colonize economically important crops such as peanuts, cotton, maize, and other oil seed crops during preharvest or storage. Its most efficient mode of dissemination is the production of airborne conidia. In addition, A. flavus produces resistant structures called sclerotia, which allow this fungus to survive adverse environmental conditions for long periods of time (1-3). This opportunistic pathogen produces a wide range of secondary metabolites, including aflatoxins (AFs). Among them, AFB1 is the most mutagenic and carcinogenic natural compound known (4-8). Ingestion of food products contaminated with AFs has been associated with hepatotoxicity, teratogenicity, immunosuppression, and liver cancer (6, 9). AF contamination also results in a negative impact on the economy in developed countries. In the United States alone A. flavus causes more than a billion dollar in losses per year due to contaminated crops (10). In addition to AFs, A. flavus is known to produce other mycotoxins, including cyclopiazonic acid (CPA), a suppressor of the calcium-dependent ATPase in the sarcoplasmic reticulum, and aflatrem, a tremogenic mycotoxin causative of neurological disorders (11,12).Studies of the A. flavus genome have revealed many gene clusters possibly connected to the synthesis of other secondary metabolites. Specifically, 55 different clusters were predicted based on the presence of genes encoding polyketide synthases (PKSs), non...

<i>Aspergillus flavus</i> Secondary Metabolites: More than Just Aflatoxins

“…Such experiments include a bottom-up approach where specific genes are targeted for disruption or ectopic expression and the resulting effect on transcriptional regulation or biological processes is determined [26][27][28][29] . Other common approaches include the use of chemical treatment, environmental conditioning, host-pathogen interaction or other stimuli relevant to the investigation to identify novel genes, proteins, interactions, expression profiles, and entire processes responding to the stimuli [30][31][32][33] .…”

“…Subsequent to this, the examination of other SM gene clusters using customized microarrays were reported 8,29,49,50) . An early effort at this was by Georgianna et al 8) whereby a collection of microarray data from 28 different treatments was used to comprehensively associate cluster expression patterns depending on treatment and environmental conditions.…”

“…A second comparison whereby A. flavus was grown on autoclaved kernels and live kernels in the field identified effects of 16 different clusters, including the pyrazinone, leporin, CPA and AF clusters. Cary et al observed the altered co-expression of clustered genes using microarray analysis of a veA knockout strain to identify the sclerotia-related clusters that synthesize aflavarin 29) and asparasone 52) . Both aflavarin (cluster 39) and asparasone (cluster 27) were among the clusters predicted to be sclerotiarelated in the Georgianna et al 8) microarray analysis and by Ehrlich and Mack 18) based on the temporal correlation of sclerotia development and cluster expression.…”

Aflatoxins: biosynthesis, prevention and eradication

“…Deletion of veA in A. flavus and A. parasiticus strains caused disruption in aflatoxin production. Hundreds of genes in A. flavus are regulated by VeA and are also influenced by the presence or absence of light, including developmental genes and secondary metabolite gene clusters (Maplestone et al, 1992;Hicks et al, 1997;Calvo et al, 2002;Bayram et al, 2008;Cary et al, 2015). The putative methyltransferase LaeA and the velvet protein VeA have been shown to interact with each other to form a protein complex designated velvet (Bayram et al, 2008;Calvo and Cary, 2015).…”

“…While A. flavus generally produces only the type B AF, other species, such as A. parasiticus and A. nomius can produce both B and G AF (Kumeda et al, 2003;Varga et al, 2003). AFB 1 , which is produced by A. flavus, is the most mutagenic and carcinogenic natural compound known (Georgianna and Payne, 2009;Cary et al, 2015). AFB 1 is converted in vivo by cytochrome P-450 mono oxygenases into reactive AFB 1 -8,9-epoxide, which binds to cellular macromolecules and causes injury to the periportal regions of the liver leading to liver cancer development (Ueng et al, 1995).…”