Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/28/2008
Publication Date: 10/29/2008
Citation: Chang, P.-K. 2008. Aspergillus parasiticus crzA, Which Encodes Calcineurin Response Zinc-Finger Protein, Is Required for Aflatoxin Production under Calcium Stress. International Journal of Molecular Sciences. 9:2027-2043. Interpretive Summary: Fungal species of Aspergillus flavus, A. parasiticus and A. nomius produce toxic and carcinogenic aflatoxins. The synthesis of these toxins is affected by nutritional factors such as carbon and nitrogen sources. Metal ions and trace elements also affect aflatoxin production. Changes in cytosolic calcium levels are known to serve as a signal which can trigger various cellular responses to stresses. A gene (crzA) critical to the tolerance of A. flavus/parasiticus to salt stress was isolated. Inactivating crzA severely impairs fungal growth and asexual development under increased calcium concentrations. The crzA gene is dispensable under normal culture conditions, but it plays a positive role in aflatoxin biosynthesis under calcium stress conditions. Prevention strategies targeting crzA could restrict fungal reproduction and dissemination and may inhibit aflatoxin production under stress conditions.
Technical Abstract: Calcium has been reported to be required for aflatoxin production. Calcium, like cAMP, is a second messenger. Cacineurin, a calmodulin-dependent serine/threonine protein phosphatase, is an important component of the calcium signaling pathway. The control of calcineurin-dependent gene expression is via the activation and specific binding of a transcription factor, Crz1, to the calcineurin-dependent response elements. Two morphologically different Aspergillus parasiticus strains, one producing aflatoxins and abundant conidia and the other accumulating O-methylsterimatocystin (OMST) and predominantly sclerotial, were used to assess the role of crzA, the yeast crz1 orthologue, in aflatoxin biosynthesis. Under normal culture conditions, the aflatoxigenic BN9'crzA mutants showed a slightly decreased radial growth, and the OMST-accumulating RH'crzA mutants showed decreased conidiation and delayed sclerotial formation. The asexual development of the RH'crzA mutants was more sensitive to increased concentrations of lithium, sodium, and potassium than that of the BN9'crzA mutants. Vegetative growth and asexual development of the 'crzA mutants of both strains were hypersensitive to increased calcium concentrations. Compared to the parental strains the production of aflatoxins or OMST of the 'crzA mutants was significantly decreased by calcium supplementation (10 mM) while allows prolific growth of vegetative mycelia. The calcium supplementation resulted in 3-fold and 2-fold decreases in the relative expression of the endoplasmic reticulum calcium ATPase 2 gene in the BN9 and RH parental strains, respectively, but changes in both 'crzA mutants were not significant. The relative expression levels of aflatoxin biosynthesis genes, nor1, ver1, and omtA, in both 'crzA mutants were decreased significantly, but the decreases in the parental strains were at much lower extents. CrzA is important in maintaining calcium homeostasis which is critical for normal growth and asexual development, and it plays a positive role in aflatoxin biosynthesis under calcium stress conditions.