Function and regulation of aflJ in the accumulation of aflatoxin early pathway intermediate in Aspergillus flavus.

aflJ resides within the aflatoxin biosynthetic gene cluster adjacent to the pathway regulatory gene aflR and is involved in aflatoxin production, but its function is unknown. Over-expression of aflJ in the aflatoxin-producing strain 86-10 resulted in increased aflatoxin. In an effort to study the function and regulation of aflJ, strain 649-1 lacking the entire biosynthetic cluster was transformed with either reporter constructs, expression constructs, or cosmid clones and analysed for gene expression or metabolite accumulation. Over-expression of aflJ did not result in elevated transcription of ver-1, omtA or aflR. To determine if over-expression of aflJ leads to an increase in early pathway intermediates, strain 649-1 was transformed with cosmid 5E6 and either gpdA::aflJ alone, gpdA::aflR alone, or aflJ and aflR together. Cosmid 5E6 contains the genes pksA, nor-1, fas-1, and fas-2, which are required for the biosynthesis of the early pathway intermediate averantin. 649-1 transformants containing 5E6 alone produced no detectable averantin. In contrast, 5E6 transformants with gpdA::aflR produced averantin, but only half as much as those transformants containing both aflR and aflJ. Northern blot analysis showed that 5E6 transformants containing both aflR and aflJ had five times more pksA transcripts and four times more nor-1 transcripts than 5E6 transformants containing gpdA::aflR alone. Further, aflJ transcription was regulated by aflR. Over-expression of aflR resulted in elevated aflJ transcription. aflJ appears to modulate the regulation of early genes in aflatoxin biosynthesis.

Elucidation of veA-dependent genes associated with aflatoxin and sclerotial production in Aspergillus flavus by functional genomics.

The aflatoxin-producing fungi, Aspergillus flavus and A. parasiticus, form structures called sclerotia that allow for survival under adverse conditions. Deletion of the veA gene in A. flavus and A. parasiticus blocks production of aflatoxin as well as sclerotial formation. We used microarray technology to identify genes differentially expressed in wild-type veA and veA mutant strains that could be involved in aflatoxin production and sclerotial development in A. flavus. The DNA microarray analysis revealed 684 genes whose expression changed significantly over time; 136 of these were differentially expressed between the two strains including 27 genes that demonstrated a significant difference in expression both between strains and over time. A group of 115 genes showed greater expression in the wild-type than in the veA mutant strain. We identified a subgroup of veA-dependent genes that exhibited time-dependent expression profiles similar to those of known aflatoxin biosynthetic genes or that were candidates for involvement in sclerotial production in the wild type.

Comparative analysis of BiP gene expression in maize endosperm.

Binding protein (BiP) is the endoplasmic reticulum member of the highly conserved HSP70 (heat shock protein 70) family of molecular chaperones. We have isolated and characterized two different BiP cDNA clones corresponding to genes expressed in immature kernels. These two cDNAs share extensive sequence similarity but map to unlinked loci in the maize genome. A comparison of the aa sequences predicted from the cDNA clones revealed only six aa differences between them. Investigation of gene-specific expression was carried out by RNA gel blot analysis. RNAs corresponding to both cDNA clones were present in increased amounts in the endosperm of floury-2 (fl2), Mucronate (Mc) and Defective endosperm-B30 (De*-B30) maize mutants, which produce abnormal storage proteins. Similar increases in RNAs corresponding to both probes were detected in cells treated with either of two agents that interfere with protein folding, azetidine-2-carboxylic acid (AZC) and tunicamycin. Investigation of the genomic complexity of the BiP genes by Southern blot analysis revealed several cross-hybridizing bands. These results are suggestive that the BiP genes expressed in endosperm are coordinately regulated members of a more complex maize BiP multigene family.

A maize ribosome-inactivating protein is controlled by the transcriptional activator Opaque-2.

Although synthesis of the cytosolic maize albumin b-32 had been shown to be controlled by the Opaque-2 regulatory locus, its function was unknown. We show here that b-32 is a member of the large and widely distributed class of toxic plant proteins with ribosome-inactivating activity. These ribosome-inactivating proteins (RIPs) are RNA N-glycosidases that remove a single base from a conserved 28S rRNA loop required for elongation factor 1 alpha binding. Cell-free in vitro translation extracts were used to show that both maize and wheat ribosomes were resistant to molar excesses of b-32 but not to the dicotyledonous RIP gelonin. We extracted RIP activity from kernels during seed maturation and germination. The amount of RIP activity increased during germination, although the amount of b-32 protein remained fairly constant. Expression of a maize RIP gene under the control of an endosperm-specific transcriptional regulatory may be an important clue prompting investigation of the biological basis for RIP expression in seeds of other plants.

Characterization of an immunoglobulin binding protein homolog in the maize floury-2 endosperm mutant.

The maize b-70 protein is an endoplasmic reticulum protein overproduced in the floury-2 (fl2) endosperm mutant. The increase in b-70 levels in fl2 plants occurs during seed maturation and is endosperm specific. We have used amino acid sequence homology to identify b-70 as a homolog of mammalian immunoglobulin binding protein (BiP). Purified b-70 fractions contain two 75-kilodalton polypeptides with pl values of 5.3 and 5.4. Both 75-kilodalton polypeptides share several properties with BiP, including the ability to bind ATP and localization within the lumen of the endoplasmic reticulum. In addition, both b-70 polypeptides can be induced in maize cell cultures with tunicamycin treatment. Like BiP, the pl 5.3 form of b-70 is post-translationally modified by phosphorylation and ADP-ribosylation. However, modification of the pl 5.4 species was not detected in vitro or in vivo. Although the b-70 gene is unlinked to fl2, b-70 overproduction is positively correlated with the fl2 gene and is regulated at the mRNA level. In contrast, the fl2 allele negatively affects the accumulation of the major endosperm storage proteins. The physical similarity of b-70 to BiP and its association with abnormal protein accumulation in fl2 endoplasmic reticulum may reflect a biological function to mediate protein folding and assembly in maize endosperm.