Roles of two new transcription factors in the synergistic effect of combined cinnamaldehyde and nonanal against Aspergillus flavus

Abstract

Aspergillus flavus, an opportunistic pathogen, is renowned for colonizing agricultural crops, leading to aflatoxin contamination. In this study, the inhibitory potency of combined cinnamaldehyde and nonanal (SCAN) against A. flavus on stored grains was evaluated, and the mechanism of action at the molecular level was explored. SCAN exhibited synergistic action against A. flavus on wheat, resulting in a 99.17% decrease in spore production. This was clearly a greater decrease than if the individual compounds were used separately. RNA-sequencing and gene knock-out analyses were performed to clarify the synergistic mechanism. Transcriptome data revealed that two new transcription factors (TFs), TF-1 (AFLA_049760) and TF-2 (AFLA_048110), were down-regulated when exposed to 0.03 μL/mL and 0.45 μL/mL SCAN. The TF-1 and TF-2 deletion mutants presented slower growth rates compared with wild-type A. flavus. In addition, conidial production was decreased by down-regulating the conidiation genes brlA and abaA. Stress assays and confocal laser scanning microscopic examinations revealed that more reactive oxygen species accumulated in ΔTF-1 mutants compared with wild-type A. flavus. These two TFs positively regulated the production of aflatoxin B1 biosynthesis and down-regulated the expression of genes, including aflM, aflQ, aflR and aflS, in the aflatoxin gene cluster. More importantly, our results indicated that the mutants were impaired in their ability to infect crop seeds. Thus, the two new TFs, which play key roles in asexual development, aflatoxin B1 biosynthesis and fungal virulence in A. flavus, may be the critical regulators in the fungus after exposure to SCAN.