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Title: Association with AflR in endosomes reveals new functions for AflJ in aflatoxin biosynthesis

item Ehrlich, Kenneth
item Mack, Brian
item Wei, Qijian - Mei Mei
item Li, Ping
item ROZE, LUDMILA - Michigan State University
item DAZZO, FRANK - Michigan State University
item Cary, Jeffrey
item Bhatnagar, Deepak
item LINZ, JOHN - Michigan State University

Submitted to: Toxins
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/12/2012
Publication Date: 12/19/2012
Citation: Ehrlich, K., Mack, B.M., Wei, Q., Li, P., Roze, L.V., Dazzo, F., Cary, J.W., Bhatnagar, D., Linz, J.E. 2012. Association with AflR in endosomes reveals new functions for AflJ in aflatoxin biosynthesis. Toxins. 4:1582-1600.

Interpretive Summary: The goal of research in the Food and Feed Safety Unit is to find effective ways to specifically inhibit aflatoxin biosynthesis in Aspergillus flavus species capable of producing this toxin in susceptible plants. Four genes involved in biosynthesis of aflatoxins by the fungus, fasA, fasB, pksA, and aflJ, have been considered to be excellent targets for inhibition because their inhibition would prevent the ability of the fungus to make aflatoxin while having no effect on the fungus’s ability to grow or reproduce. One of these, pksA, is defective in the non-aflatoxin producing strain now currently being used for biocontrol of aflatoxin in cotton, AF36. In the present study we show for the first time that one of these genes, aflJ, is necessary for aflatoxin production because it is required for formation of the biosynthetic vesicle (part of the cell) in which the biosynthesis occurs. Without this gene and its protein, there is no biosynthesis of aflatoxin. Our data show that this protein is bound to AflR, the transcription regulatory protein in the vesicle and is necessary for movement of AflR to the nucleus in order for AflR to activate production of the RNA needed for biosynthesis. This insight into the function of AflR is crucial to the goal of developing a specific inhibitor of AflJ function.

Technical Abstract: Aflatoxins are the most potent naturally occurring carcinogens of fungal origin. Biosynthesis of aflatoxin involves the coordinated expression of more than 25 genes. The function of one gene in the aflatoxin gene cluster, aflJ, is not entirely understood but, because previous studies demonstrated a physical interaction between the Zn2Cys6 transcription factor AflR and AflJ, AflJ was proposed to act as a transcriptional co-activator. Image analysis revealed that, in the absence of aflJ in A. parasiticus, endosomes cluster within cells and near septa. AflJ fused to yellow fluorescent protein complemented the mutation in A. parasiticus 'aflJ and localized mainly in endosomes. We found that AflJ co-localizes with AflR both in endosomes and in nuclei. Chromatin immunoprecipitation did not detect AflJ binding at known AflR DNA recognition sites suggesting that AflJ either does not bind to these sites or binds to them transiently. Based on these data, we hypothesize that AflJ assists in AflR transport to or from the nucleus, thus controlling the availability of AflR for transcriptional activation of aflatoxin biosynthesis cluster genes. AflJ may also assist in directing endosomes to the cytoplasmic membrane for aflatoxin export.