The Aspergillus parasiticus protein AFLJ interacts with the aflatoxin pathway-specific regulator AFLR

Abstract

The Aspergillus parasiticus aflJgene, which is located in the aflatoxin biosynthetic gene cluster and is transcribed divergently from the aflatoxin pathway regulatory gene aflR, encodes a 438-amino acid protein. Transformation with aflJplus aflR, but not aflJalone, increased the accumulation of aflatoxin precursors substantially in an O-methylsterigmatocystin-accumulating A. parasiticus strain. Disruption of aflJ resulted in non-pigmented mutants that lost the ability to synthesize aflatoxin intermediates. Transcript profiling by real time RT-PCR indicated that the lack of aflJtranscripts in the aflJ knockout mutants is associated with a significant decrease in the transcript levels of the genes for early (pksAand nor1), middle (ver1) and later (omtA) steps in the aflatoxin biosynthetic pathway, with the degree of reduction ranging from 5- to 20-fold. Deletion of aflJ, however, did not have any effect on the aflRtranscript level, and vice versa. Two-hybrid assays showed that AFLJ did not interact with aflatoxin biosynthetic enzymes, including NOR1, VER1, OMTA and ORDA. But AFLJ interacted with full-length AFLR, and the DNA-binding domain of AFLR was found not to be essential for this interaction. Simultaneous substitution of Arg427, Arg429, and Arg431 in the C-terminal region of AFLR with Leu residues abolished its ability to interact with AFLJ. Replacement of Asp436, which was previously shown to be crucial for AFLR's activation activity, with His, in contrast, had little effect on the interaction. On the other hand, deletions in most regions of AFLJ appeared to destroy its function, despite the fact that random amino acid substitution(s) in its C-terminal region did not drastically affect its capacity to interact with AFLR. The results show that aflJis involved in the expression of aflatoxin structural genes and support the hypothesis that aflJis a coactivator gene.