Regulation of nitrogen utilization and mycotoxin biosynthesis by the GATA transcription factor AaAreA in Alternaria alternata

Alternaria alternata is a prevalent postharvest pathogen that generates diverse mycotoxins, notably alternariol (AOH) and alternariol monomethyl ether (AME), which are recurrent severe contaminants. Nitrogen sources modulate fungal growth, development, and secondary metabolism, including mycotoxin production. The GATA transcription factor AreA regulates nitrogen source utilization. However, little is known about its involvement in the regulation of nitrogen utilization in A. alternata. To examine the regulatory mechanism of AaAreA on AOH and AME biosynthesis in A. alternata, we analyzed the impact of diverse nitrogen sources on the fungal growth, conidiation and mycotoxin production. The use of a secondary nitrogen source (NaNO₃) enhanced mycelial elongation and sporulation more than the use of a primary source (NH₄Cl). NaNO₃ favored greater mycotoxin accumulation than did NH₄Cl. The regulatory roles of AaAreA were further clarified through gene knockout. The absence of AaAreA led to an overall reduction in growth in minimal media containing any nitrogen source except NH₄Cl. AaAreA positively regulates mycotoxin biosynthesis when both NH₄Cl and NaNO₃ are used as nitrogen sources. Subcellular localization analysis revealed abundant nuclear transport when NaNO₃ was the sole nitrogen source. The regulatory pathway of AaAreA was systematically revealed through comprehensive transcriptomic analyses. The deletion of AaAreA significantly impedes the transcription of mycotoxin biosynthetic genes, including aohR, pksI and omtI. The interaction between AaAreA and aohR, a pathway-specific transcription factor gene, demonstrated that AaAreA binds to the aohR promoter sequence (5'-GGCTATGGAAA-3'), activating its transcription. The expressed AohR regulates the expression of downstream synthase genes in the cluster, ultimately impacting mycotoxin production. This study provides valuable information to further understand how AreA regulates AOH and AME biosynthesis in A. alternata, thereby enabling the effective design of control measures for mycotoxin contamination.