Aflavarin

The 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.

In filamentous fungi, homeobox proteins are conserved transcriptional regulators described to control conidiogenesis and fruiting body formation. Eight homeobox (hbx) genes are found in the genome of the aflatoxin-producing ascomycete, Aspergillus flavus While loss-of-function of seven of the eight genes had little to no effect on fungal growth and development, disruption of hbx1, resulted in aconidial colonies and lack of sclerotial production. Furthermore, the hbx1 mutant was unable to produce aflatoxins B1 and B2, cyclopiazonic acid and aflatrem. In the present study, hbx1 transcriptome analysis revealed that hbx1 has a broad effect on A. flavus gene expression, and the effect of hbx1 increases overtime, impacting more than five thousand protein-coding genes. Among the affected genes, those in the category of secondary metabolism (SM), followed by that of cellular transport, were the most affected. Specifically, regarding the effect of hbx1 on SM, we found that genes in 44 SM gene clusters where upregulated while 49 were downregulated in the absence of hbx1, including genes in the SM clusters responsible for the synthesis of asparasone, piperazine and aflavarin, all known to be associated with sclerotia. In addition, our study revealed that hbx1 affects the expression of other transcription factor genes involved in development, including the conidiation central regulatory pathway and flb genes.

Aspergillus flavus aswA (AFLA_085170) is a gene encoding a Zn(II)2Cys6 DNA-binding domain and a transcriptional activation domain, DUF3468. Disruption of aswA yielded strains that made a truncated gene transcript and generated a fungus that produced a greatly increased number of sclerotia. These sclerotia were odd-shaped and non-pigmented (white) and different from oval and pigmented (dark brown to black) mature sclerotia. Transcriptomic analysis of the ΔaswA strain grown on potato dextrose agar plates and Wickerham agar plates showed that expression of clustering genes involved in the biosynthesis of three sclerotium-associated secondary metabolites was down-regulated. These included gene clusters of asparasone, aflatrem, and aflavarin. In contrast, those of aflatoxin, cyclopiazonic acid and kojic acid were not affected. Metabolite analyses confirmed that the non-pigmented sclerotia contained aflatoxin and cyclopiazonic acid but not other aforementioned metabolites, three asparasone analogs and dihydroxyaflavinine commonly present in mature sclerotia. Impairment in aswA gene function stalls normal sclerotial development, which in turn prevents biosynthesis and accumulation of sclerotium-specific metabolites.