Transcriptome Sequencing Revealed an Inhibitory Mechanism of Recombinant Puroindoline B Protein on Aspergillus flavus

<i>Aspergillus flavus</i>, a common food contaminant, poses health and economic risks. Previous research showed that recombinant Puroindoline B protein (rPINB) inhibited <i>A. flavus</i> by disrupting its cell wall, membrane, nuclear function, mitochondrial activity, and oxidative stress. This study used transcriptome technology to explore the impact of rPINB on <i>A. flavus</i> gene expression and created gene deletion strains to test the sensitivity to rPINB. RNA-Seq identified the differentially expressed genes (DEGs) affecting cell wall synthesis, membrane transport, oxidative stress, spore formation, and aflatoxin production. The MFS transporter genes AFLA_106900 (<i>mfs1</i>) and AFLA_106910 (<i>mfs2</i>) were crucial for an inhibitory effect of rPINB. The mutants exhibited reduced sensitivity to rPINB-mediated inhibition, indicating lower growth, sunken conidia, and shriveled hyphae, compared to the wild-type strain. The results also demonstrated decreased sensitivity to the stress agents affecting cell membranes, osmotic balance, and oxidation, alongside a significant reduction in AFB1 production in gene-deleted strains. These results suggested that <i>mfs1</i> and <i>mfs2</i> were essential for rPINB protein's inhibition of <i>A. flavus</i> growth, laying the groundwork for the mold control strategies using plant proteins.