Submitted to: Toxins
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 25, 2011
Publication Date: August 2, 2011
Citation: Wilkinson, J.R., Kale, S.P., Bhatnagar, D., Yu, J., Ehrlich, K. 2011. Expression profiling of non-aflatoxigenic Aspergillus parasiticus mutants obtained by 5-azacytosine treatment or serial mycelial transfer. Toxins. 3(8):932-948. Interpretive Summary: With the availability of the complete genome sequence and the sequences of all proteins made by the fungus Aspergillus flavus it is possible to better understand what factors in the environment “turn on” aflatoxin production by this mold. Aflatoxin is highly toxic and cancer-causing product made by the fungus under certain environmental conditions and only on some crops. A new method for comparing mutants of A. flavus (which cannot make the toxin) to the parental fungus (which makes the toxin) uses what is called microarray analysis. This involves comparing the mRNA (the nucleic acid that codes for the proteins) in the parent strain of fungus to the mRNA made by the mutants and determining which mRNAs are made in greater or lesser amounts. In this paper we found that even though the proteins for making aflatoxin are not affected by the mutations, mRNAs coding for proteins necessary for formation of a cell organelle (a small organ within the fungal cell) called a peroxisome are significantly lower in the mutant fungi. This means that it may be possible to prevent aflatoxin production by exposing the fungus to chemicals that prevent formation of this cellular component. It also can explain why the ability to make aflatoxins by the A. flavus is restricted to certain plants, because the plants themselves may make inhibitors.
Technical Abstract: Aflatoxins are carcinogenic secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus. Repeated serial mycelial transfer or treatment of A. parasiticus with 5-azacytidine produced mutants with a fluffy phenotype and loss of aflatoxin production. To understand how these treatments affect aflatoxin production and development, we carried out expressed sequence tag (EST)-based microarray assays to identify differentially expressed genes in clones obtained from these treatments. Expression of 183 genes was significantly dysregulated. Of these, 38 had at least two-fold or lower expression compared to the untreated control and only two had two-fold or higher expression. The most frequent change was downregulation of genes predicted to be membrane-bound. Dysregulation of some of these may be responsible for the fluffy phenotype. Of the aflatoxin biosynthesis pathway genes only aflJ (aflS) was significantly affected by either treatment. A gene for a protein homologous to a key regulator of secondary metabolite biosynthesis (LaeA) was one of the upregulated genes and possibly could affect the activity of LaeA. Other genes known to be required for fungal developmental or aflatoxin production were not affected by the treatments. Consistent with the fluffy phenotype and the non-aflatoxigenicity of the clones obtained by either treatment, we hypothesize that the mutations cause improper development of conidiophores and specialized biosynthesis vacuoles (aflatoxisomes) needed for AF production.