Aspergillus flavus GPI-anchored protein-encoding ecm33 has a role in growth, development, aflatoxin biosynthesis, and maize infection

Perng-Kuang Chang, Qi Zhang, Leslie Scharfenstein, Brian Mack, Akira Yoshimi, Ken Miyazawa, Keietsu Abe

Many glycosylphosphatidylinositol-anchored proteins (GPI-APs) of fungi are membrane enzymes, organization components, and extracellular matrix adhesins. We analyzed eight Aspergillus flavus transcriptome sets for the GPI-AP gene family and identified AFLA_040110, AFLA_063860, and AFLA_113120 to be among the top 5 highly expressed genes of the 36 family genes analyzed. Disruption of the former two genes did not drastically affect A. flavus growth and development. In contrast, disruption of AFLA_113120, an orthologue of Saccharomyces cerevisiae ECM33, caused a significant decrease in vegetative growth and conidiation, promoted sclerotial production, and altered conidial pigmentation. The A. flavus ecm33 null mutant, compared with the wild type and the complemented strain, produced predominantly aflatoxin B2 but accumulated comparable amounts of cyclopiazonic acid. It showed decreased sensitivity to Congo red at low concentrations (25-50 μg/mL) but had increased sensitivity to calcofluor white at high concentrations (250-500 μg/mL). Analyses of cell wall carbohydrates indicated that the α-glucan content was decreased significantly (p < 0.05), but the contents of chitin and ß-glucan were increased in the mutant strain. In a maize colonization study, the mutant was shown to be impaired in its infectivity and produced 3- to 4-fold lower amounts of conidia than the wild type and the complemented strain. A. flavus Ecm33 is required for proper cell wall composition and plays an important role in normal fungal growth and development, aflatoxin biosynthesis, and seed colonization.