Submitted to: Molecular Genetics and Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 30, 2002
Publication Date: March 1, 2003
Citation: Chang, P.-K. 2003. The Aspergillus parasiticus protein AFLJ interacts with the aflatoxin pathway-specific regulator AFLR. Molecular Genetics and Genomics. 268:711-719. Interpretive Summary: The molds, Aspergillus flavus and Aspergillus parasiticus, produce aflatoxin, one of the most potent liver carcinogens known. This toxin commonly contaminates a variety of agricultural commodities in the field (cotton, corn, peanuts, and some treenuts) and many other commodities in storage, thus resulting in their becoming unsalable. The synthesis of aflatoxin is a very complex process and involves more than 20 chemical steps. In this study, using molecular and genetic analyses, it was confirmed that besides the previously identified regulatory protein, AFLR, another protein, AFLJ, also controls the synthesis of aflatoxin through interacting with AFLR. Genetic interference of the interaction of these two proteins destroys the molds' ability to make aflatoxin. This observation could help to achieve the goal of reduction of preharvest aflatoxin contamination by using breeding approaches to screen for plant/crop factors that affect synthesis of these two proteins, or which interfere with the interaction. Resultant technology could reduce aflatoxin contamination levels in crops, thus saving U.S. industry millions of dollars in lost revenues.
Technical Abstract: The Aspergillus parasiticus aflJ gene, located in the aflatoxin biosynthetic gene cluster and divergently transcribed from the aflatoxin pathway regulatory gene aflR, encodes a 438-amino acid protein. Transformation of aflJ plus aflR, but not aflJ alone, 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 a lack of the aflJ transcript in the aflJ knockout mutants significantly decreased the transcript levels of the genes of the early (pksA and nor1), middle (ver1) and later (omtA) steps of aflatoxin biosynthetic pathway, respectively, and the reduction ranged from 5 to 20-fold. Deletion of aflJ, however, did not correlate with changes in the aflR transcript 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 not essential for the interaction. Simultaneous substitutions of Arg427, Arg429, and Arg431 at the carboxyl terminus of AFLR with Leu abolished its interaction with AFLJ. Substitution of Asp436 with His, previously shown to be crucial for AFLR's activation activity had little effect on the interaction. Deletions in most regions of AFLJ appear to destroy its function despite that random amino acid substitution(s) at its carboxyl terminus did not drastically affect its capacity to interact with AFLR. The results show that aflJ is involved in the expression of aflatoxin structural genes and support the hypothesis that aflJ is a coactivator gene.