New Insights of Transcriptional Regulator AflR in Aspergillus flavus Physiology

Abstract

Aspergillus flavus <i>aflR</i>, a gene encoding a Zn(II)₂Cys₆ DNA-binding domain, is an important transcriptional regulator of the aflatoxin biosynthesis gene cluster. Our previous results of Gene ontology (GO) analysis for the binding sites of AflR in A. flavus suggest that AflR may play an integrative regulatory role. In this study the Δ<i>aflR</i> and overexpression (OE) strains based on the well-established double-crossover recombinational technique were constructed to investigate the integrative function of the <i>aflR</i> gene in A. flavus. The disruption of <i>aflR</i> severely affected the aflatoxin biosynthetic pathway, resulting in a significant decrease in aflatoxin production. The aflatoxin B₁ (AFB₁) of the Δ<i>aflR</i> strain was 180 ng/mL and aflatoxin B₂ (AFB₂) was 2.95 ng/mL on YES medium for 5 days, which was 1/1,000 of that produced by the wild-type strain (WT). In addition, the Δ<i>aflR</i> strain produced relatively sparse conidia and a very small number of sclerotia. On the seventh day, the sclerotia yield on each plate of the WT and OE strains exceeded 1,000, while the sclerotial formation of the Δ<i>aflR</i> strain was not detected until 14 days. However, the biosynthesis of cyclopiazonic acid (CPA) was not affected by <i>aflR</i> gene disruption. Transcriptomic analysis of the Δ<i>aflR</i> strain grown on potato dextrose agar (PDA) plates at 0 h, 24 h, and 72 h showed that expression of clustering genes involved in the biosynthesis of aflatoxin was significantly downregulated. Meanwhile, the Δ<i>aflR</i> strain compared with the WT strain showed significant expression differences in genes involved in spore germination, sclerotial development, and carbohydrate metabolism compared to the WT. The results demonstrated that the A. flavus <i>aflR</i> gene also played a positive role in the fungal growth and development in addition to aflatoxin biosynthesis. <b>IMPORTANCE</b> Past studies of the A. flavus <i>aflR</i> gene and its orthologues in related Aspergillus species were solely focused on their roles in secondary metabolism. In this study, we used the Δ<i>aflR</i> and OE strains to demonstrate the role of <i>aflR</i> in growth and development of A. flavus. For the first time, we confirmed that the Δ<i>aflR</i> strain also was defective in production of conidia and sclerotia, asexual propagules of A. flavus. Our transcriptomic analysis further showed that genes involved in spore germination, sclerotial development, aflatoxin biosynssssthesis, and carbohydrate metabolism exhibited significant differences in the Δ<i>aflR</i> strain compared with the WT strain. Our study indicates that AflR not only plays an important role in regulating aflatoxin synthesis but also in playing a positive role in the conidial formation and sclerotial development in A. flavus. This study reveals the critical and positive role of the <i>aflR</i> gene in fungal growth and development, and provides a theoretical basis for the genetic studies of other <i>aspergilli</i>.