Identification of a copper-transporting ATPase involved in biosynthesis of A. flavus conidial pigment

Authors: Chang, Perng Kuang; Scharfenstein, Leslie; Mack, Brian; Wei, Qijian - Mei Mei; Gilbert, Matthew; Lebar, Matthew; Cary, Jeffrey

Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/31/2019
Publication Date: 4/29/2019
Citation: Chang, P.-K., Scharfenstein, L.L., Mack, B.M., Wei, Q., Gilbert, M.K., Lebar, M.D., Cary, J.W. 2019. Identification of a copper-transporting ATPase involved in biosynthesis of A. flavus conidial pigment. Applied Microbiology and Biotechnology. 103:4889-4897. https://doi.org/10.1007/s00253-019-09820-0.
DOI: https://doi.org/10.1007/s00253-019-09820-0

Interpretive Summary: Fungi commonly produced spores with characteristic pigmentation. The saprophytic fungus Aspergillus flavus, which is able to produce aflatoxin and many other secondary metabolites, produces yellowish green spores that are resistant to ultraviolet. In this study, we isolated a yellow spore mutant and determined its underlying molecular defect. In addition, we generated a gold spore mutant by gene knockout. Together with a previously available dark-green spore mutant, we determined pigment formation of wild-type A. flavus spores is in the order of yellow, gold, dark green and yellowish green. Using inhibitors that block melanin biosynthesis, we found two commonly recognized melanin biosynthesis pathways are not involved in the production of spore pigments of A. flavus.

Technical Abstract: Conidia are asexual spores and play a crucial role in fungal dissemination. Conidial pigmentation is important for tolerance against UV radiation and contributes to survival of fungi. The molecular basis of conidial pigmentation has been studied in several fungal species. In spite of sharing the initial common step of polyketide formation, other steps for pigment biosynthesis appear to be species-dependent. In this study, we isolated an Aspergillus flavus spontaneous mutant that produced yellow conidia. The underlying genetic defect, a three-nucleotide in-frame deletion in the gene, AFLA_051390, that encodes a copper-transporting ATPase, was identified by a comparative genomics approach. This genetic association was confirmed by disruption of the wild-type gene. When yellow mutants were grown on medium supplemented with copper ions or chloride ions, green conidial color was partially and nearly completely restored, respectively. Further disruption of AFLA_045660, an orthologue of Aspergillus nidulans yA (yellow pigment) that encodes a multicopper oxidase, in wild type and a derived strain producing dark green conidia showed that it yielded mutants that produced gold conidia. The results placed formation of the gold pigment after that of the yellow pigment and before that of the dark green pigment. Using reported inhibitors of DHN-melanin (tricyclazole and phthalide) and DOPA-melanin (tropolone and kojic acid) pathways on a set of conidial color mutants we investigated the involvement of melanin biosynthesis in A. flavus conidial pigment formation. Tricyclazole and phthalide did not change the conidial pigmentation of all mutants and the wild type. In contrast, tropolone was fungicidal at low concentrations (= 50µg/mL) and kojic acid inhibited conidial pigment production of all A. flavus strains including the wild type only at a high concentration (1 mg/mL) resulting in nonpigmented (white) conidia. We concluded that both pathways have no bearing on conidial pigment biosynthesis of A. flavus.