Regulation of Conidiogenesis in Aspergillus flavus

Abstract

Aspergillus flavus is a representative fungal species in the Aspergillus section Flavi and has been used as a model system to gain insights into fungal development and toxin production. A. flavus has several adverse effects on humans, including the production of the most carcinogenic mycotoxin aflatoxins and causing aspergillosis in immune-compromised patients. In addition, A. flavus infection of crops results in economic losses due to yield loss and aflatoxin contamination. A. flavus is a saprophytic fungus that disperses in the ecosystem mainly by producing asexual spores (conidia), which also provide long-term survival in the harsh environmental conditions. Conidia are composed of the rodlet layer, cell wall, and melanin and are produced from an asexual specialized structure called the conidiophore. The production of conidiophores is tightly regulated by various regulators, including the central regulatory cascade composed of BrlA-AbaA-WetA, the fungi-specific velvet regulators, upstream regulators, and developmental repressors. In this review, we summarize the findings of a series of recent studies related to asexual development in A. flavus and provide insights for a better understanding of other fungal species in the section Flavi.

Keywords: AbaA; Aspergillus flavus; BrlA; WetA; asexual development; velvet.

Figure 1

Asexual developmental regulators in Aspergillus spp. (A) A morphogenic model of conidiophore development of A. flavus. (B) A genetic model of the regulation of conidiogenesis in A. flavus. (C) Distribution of regulators involved in conidiogenesis in Aspergillus section Flavi. Distribution of 34 important regulators were investigated in 24 representative genomes from the section Flavi. The genomic data of A. albertensis, A. alliaceus CBS 536.65, A. arachidicola, A. avenaceus IBT 18842, A. bertholletius IBT 29228, A. bombycis NRRL 26010, A. caelatus CBS 763.97, A. coremiiformis CBS 553.77, A. flavus NRRL3357, A. leporis CBS 151.66, A. minisclerotigenes CBS 117635, A. nomius IBT 12657, A. novoparasiticus CBS 126849, A. oryzae RIB40, A. parasiticus CBS 117618, A. parvisclerotigenus CBS 121.62, A. pseudocaelatus CBS 117616, A. pseudonomius CBS 119388, A. pseudotamarii CBS 117625, A. sergii CBS 130017, A. tamarii CBS 117626, and A. transmontanensis CBS 130015 are obtained from Joint Genome Institute fungal genome portal MycoCosm (http://jgi.doe.gov/fungi, accessed on 4 August 2022). The genomic data of A. hancockii and A. sojae SMF134 were previously published [29,30]. The homologs were searched by BlastP using the regulators of A. flavus NRRL3357 as queries.

Figure 2

Summary of the central regulators in A. flavus. (A) Phenotypes of ΔbrlA, ΔabaA, and ΔwetA mutant strains. Colony phenotypes of ΔbrlA, ΔabaA, and ΔwetA strains point-inoculated on solid glucose minimal medium with 0.1% yeast extract media and grown at 37 °C (Upper). Morphology of ΔbrlA, ΔabaA, and ΔwetA conidiophores observed under a light microscope at 48 h after inoculation onto solid MMYE media at 37 °C (Bottom). (B) Scanning electron micrographs of ΔbrlA and ΔabaA strains. (C) mRNA levels of brlA, abaA, and wetA during A. flavus life cycle. Samples for RT-qPCR analysis were collected from 12, 18, and 24 h of vegetative growth; 6, 12, 24, and 48 h of asexual development, and in conidia.

Figure 3

Roles of the central regulators in A. flavus sclerotia and aflatoxin production. (A) Ethanol-washed colony photographs of ΔbrlA, ΔabaA, and ΔwetA strains grown on solid minimal media with 1% glucose and 0.1% yeast extract (MMYE) for 7 days. Quantitative analysis of sclerotia of these strains. (B) Image of thin-layer chromatography (TLC) of aflatoxin B1 from ΔbrlA, ΔabaA, and ΔwetA strains under dark conditions. To extract aflatoxin B1 from each strain, about 10⁷ conidia were inoculated into liquid complete media and incubated for 7 days at 30°C in dark condition. To extract aflatoxin B1, chloroform was used. The samples were spotted onto a TLC silica plate, and the plate was placed into a chamber containing chloroform: acetone (9:1, v/v). Densitometry of the TLC analysis results. Statistical differences between control and mutant strains were evaluated using Student’s unpaired t-tests. Data are reported as the mean ± standard deviation. ** p < 0.01, *** p < 0.001.

Figure 4

Roles of the fluffy genes in A. flavus. (A) Colony morphology of ΔflbB, ΔflbC, ΔflbD, and ΔflbE mutant strains. (B) Quantitative analysis of the number of conidia per plate shown in (A). (C) mRNA level of brlA in wild-type and mutant strains. (D) Ethanol-washed colony photographs of fluffy mutant strains. (E) Image of TLC of aflatoxin B1 from fluffy mutant strains. Densitometry of TLC analysis results. Statistical differences between control and mutant strains were evaluated using Student’s unpaired t-tests. Data are reported as the mean ± standard deviation. * p < 0.05, ** p < 0.01, and *** p < 0.001.