Bioinformatics and analysis of differentially expressed genes (DEGs)

Raw RNA-seq data (FASTQ format) were processed with fastp, which removes adapter and poly-N sequences, yielding clean data/reads³⁵, and the Q20, Q30 and GC content of clean data were calculated. All downstream analyses were based on high-quality, clean data. Paired-end, clean reads were mapped to the A. flavus NRRL 3357 genome (EnsemblFungi genome assembly JCVI-afl1-v2.0; www.ebi.ac.uk/ena/browser/view/GCA_000006275.2) using HISAT2 software³⁶. Quantification of gene expression was calculated according to Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced (FPKM)³⁷; FPKM ≥ 1 was set as the threshold for an expressed gene. Differentially expressed genes (DEGs) between control and test samples were analyzed using the DESeq2 R package³⁸. The combination of an FPKM fold-change ≥ 2 (or |log2 (fold change)| ≥ 1) and adjusted p-value < 0.05 was set as the threshold for DEGs in FunCat³⁹ analysis performed using FungiFun⁴⁰.