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Hypocrealean entomopathogenic fungi produce a large variety of secondary metabolites with diverse ecological roles. These compounds serve as nutraceuticals and traditional remedies, and as drug leads for the modern pharmaceutical industry.

Reversible protein phosphorylation is known to play important roles in the regulation of various cellular processes in eukaryotes. Phosphatase-mediated dephosphorylation are integral components of cellular signal pathways by counteracting the phosphorylation action of kinases. In this study, we characterized the functions of CDC14, a dual-specificity phosphatase in the development, secondary metabolism and crop infection of Aspergillus flavus. Deletion of AflCDC14 resulted in a growth defect and abnormal conidium morphology. Inactivation of AflCDC14 caused defective septum and failure to generate sclerotia. Additionally, the AflCDC14 deletion mutant (ΔCDC14) displayed increased sensitivity to osmotic and cell wall integrity stresses. Importantly, it had a significant increase in aflatoxin production, which was consistent with the up-regulation of the expression levels of aflatoxin biosynthesis related genes in ΔCDC14 mutant. Furthermore, seeds infection assays suggested that AflCDC14 was crucial for virulence of A. flavus. It was also found that the activity of amylase was decreased in ΔCDC14 mutant. AflCDC14-eRFP mainly localized to the cytoplasm and vesicles during coidial germination and mycelial development stages. Taken together, these results not only reveal the importance of the CDC14 phosphatase in the regulation of development, aflatoxin biosynthesis and virulence in A. flavus, but may also provide a potential target for controlling crop infections of this fungal pathogen.

In Aspergillus, the cyclic adenosine monophosphate (cAMP) signaling modulates asexual development and mycotoxin biosynthesis. Here, we characterize the cyclase-associated protein Cap in the pathogenic fungus Aspergillus flauvs. The cap disruption mutant exhibited dramatic reduction in hyphal growth, conidiation, and spore germination, while an enhanced production of the sclerotia was observed in this mutant. Importantly, the cap gene was found to be important for mycotoxin biosynthesis and virulence. The domain deletion study demonstrated that each domain played an important role for the Cap protein in regulating cAMP/protein kinase A (PKA) signaling, while only P1 and CARP domains were essential for the full function of Cap. The phosphorylation of Cap at S35 was identified in A. flavus, which was found to play a negligible role for the function of Cap. Overall, our results indicated that Cap with multiple domains engages in mycotoxin production and fungal pathogenicity, which could be designed as potential control targets for preventing this fungal pathogen.

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Aspergillus flavus is a renowned plant, animal and human pathogen. areA is a global nitrogen regulatory gene of the GATA transcription factor family, shown to be the major nitrogen regulator. In this study, we identified areA in A. flavus and studied its function. The AreA protein contained a signatory zinc finger domain, which is extremely conserved across fungal species. Gene deletion (∆areA) and over-expression (OE::areA) strains were constructed by homologous recombination to elucidate the role of areA in A. flavus. The ∆areA strain was unable to efficiently utilize secondary nitrogen sources for growth of A. flavus, and it had poorly developed conidiophores, when observed on complete medium, resulting in the production of significantly less conidia than the wild-type strain (WT). Aflatoxin B1 (AFB1) production was reduced in ∆areA compared with the WT strain in most conditions tested, and ∆areA had impaired virulence in peanut seeds. areA also played important roles in the sensitivity of A. flavus to osmotic, cell wall and oxidative stresses. Hence, areA was found to be important for the growth, aflatoxin production and pathogenicity of A. flavus. This work sheds light on the function of areA in the regulation of the nitrogen metabolism of A. flavus, and consequently aims at providing new ways for controlling the crossover pathogen, A. flavus. Key Contribution:The GATA transcription factor and major nitrogen regulator AreA, was shown to regulate nitrogen metabolism and aflatoxin B1 production in A. flavus.

Fungal secondary metabolites play important roles not only in fungal ecology but also in humans living as beneficial medicine or harmful toxins. In filamentous fungi, bZIP-type transcription factors (TFs) are associated with the proteins involved in oxidative stress response and secondary metabolism. In this study, a connection between a bZIP TF and oxidative stress induction of secondary metabolism is uncovered in an opportunistic pathogen Aspergillus flavus, which produces carcinogenic and mutagenic aflatoxins. The bZIP transcription factor AflRsmA was identified by a homology research of A. flavus genome with the bZIP protein RsmA, involved in secondary metabolites production in Aspergillus nidulans. The AflrsmA deletion strain (∆AflrsmA) displayed less sensitivity to the oxidative reagents tert-Butyl hydroperoxide (tBOOH) in comparison with wild type (WT) and AflrsmA overexpression strain (AflrsmA OE ), while AflrsmA OE strain increased sensitivity to the oxidative reagents menadione sodium bisulfite (MSB) compared to WT and ∆AflrsmA strains. Without oxidative treatment, aflatoxin B 1 (AFB 1 ) production of ∆AflrsmA strains was consistent with that of WT, but AflrsmA OE strain produced more AFB 1 than WT; tBOOH and MSB treatment decreased AFB 1 production of ∆AflrsmA compared to WT. Besides, relative to WT, ∆AflrsmA strain decreased sclerotia, while AflrsmA OE strain increased sclerotia. The decrease of AFB 1 by ∆AflrsmA but increase of AFB 1 by AflrsmA OE was on corn. Our results suggest that AFB 1 biosynthesis is regulated by AflRsmA by oxidative stress pathways and provide insights into a possible function of AflRsmA in mediating AFB 1 biosynthesis response host defense in pathogen A. flavus.Key Contribution: Without oxidative treatment, deletion of AflrsmA has no impact on aflatoxin B 1 (AFB 1 ) biosynthesis in comparison with the wild type strain. However, the AflrsmA disruptant produces less AFB 1 on corn seeds and on YGT medium containing oxidative reagent menadione sodium bisulfite or tert-Butyl hydroperoxide.Toxins 2020, 12, 271 2 of 17 basic leucine zipper (bZIP) domain. The bZIP-type transcription factor as one of the largest families of dimerizing TFs is widely distributed in the all eukaryotes genomes. The first bZIP-type TF was discovered in humans over 30 years ago [2]. Then, it was found that bZIP-type TFs are involved in metabolism, development, cell cycle, reproduction, and programmed cell death [1]. In plants, the functions of bZIP-type TFs include abiotic stress response, metabolism, mediating pathogen defense, hormone signaling, and senescence [3]. bZIP-type TFs are involved in various biological processes in fungi (described below).In Saccharomyces cerevisiae, the first bZIP-type TF called Yap (yeast activator protein) was found [4], then a subset of YAP TFs (YAP1-YAP8) have been well defined [5]. Several bZIP proteins have been reported in filamentous fungi, such as Aspergillus spp. [6-11], Neurospora crassa [12], plant pathogens Fusarium graminearum [13], Magnaporthe oryzae [14], and ...