Abstract

   Aspergillus flavus is a soilborne pathogenic fungus that poses a
   serious public health threat due to it contamination of food with
   carcinogenic aflatoxins. Our previous studies have demonstrated that
   benzenamine displayed strong inhibitory effects on the mycelial growth
   of A. flavus. In this study, we systematically investigated the
   inhibitory effects of benzenamine on the development, aflatoxin
   biosynthesis, and virulence in A. flavus, as well as the underlying
   mechanism. The results indicated that benzenamine exhibited great
   capacity to combat A. flavus at a concentration of 100 µL/L, leading to
   significantly decreased aflatoxin accumulation and colonization
   capacity in maize. The transcriptional profile revealed that 3589 genes
   show altered mRNA levels in the A. flavus after treatment with
   benzenamine, including 1890 down-regulated and 1699 up-regulated genes.
   Most of the differentially expressed genes participated in the
   biosynthesis and metabolism of amino acid, purine metabolism, and
   protein processing in endoplasmic reticulum. Additionally, the results
   brought us to a suggestion that benzenamine affects the development,
   aflatoxin biosynthesis, and pathogenicity of A. flavus via
   down-regulating related genes by depressing the expression of the
   global regulatory factor leaA. Overall, this study indicates that
   benzenamine have tremendous potential to act as a fumigant against
   pathogenic A. flavus. Furthermore, this work offers valuable
   information regarding the underlying antifungal mechanism of
   benzenamine against A. flavus at the level of transcription, and these
   potential targets may be conducive in developing new strategies for
   preventing aflatoxin contamination.

   Keywords: Aspergillus flavus, aflatoxin B1, benzenamine, fumigation,
   transcriptome

1. Introduction

   Aspergillus flavus, an opportunistic pathogen of both humans and
   plants, produces an abundance of diverse secondary metabolites,
   including aflatoxins. Aflatoxins are the most important mycotoxin due
   to their common occurrence among the serious threats that are posed to
   humans and animals. About 18 different types of aflatoxin are now known
   [[40]1]. Among these, aflatoxin B1 is regarded as the most potent
   natural carcinogen and it is classified as a Group I carcinogen by the
   International Agency for Research on Cancer (IARC) [[41]2]. It is
   estimated that up to 28% of all hepatocellular carcinoma cases
   worldwide may be caused by aflatoxins [[42]3].

   A. flavus is the primary etiological agent of aflatoxin contamination
   of agricultural commodities, such as corn and peanut [[43]4]. The Food
   and Agriculture Organization (FAO) forecasts that approximately 2595
   million tonnes of cereals will be produced and 2649 million tonnes will
   be consumed in 2018 [[44]5]. In addition, cereal losses due to other
   factors, including climate-related natural disasters and conflict, have
   increased the prevalence of undernourishment. The estimated number of
   undernourished people increased to nearly 821 million in 2017 [[45]6].
   Therefore, preventing aflatoxin contamination is necessary in
   addressing the problem of food shortage and food safety.

   To minimize the harmful effects of aflatoxins, several strategies have
   been developed to control toxigenic fungus growth and aflatoxin
   production. Volatiles, such as aldehyde, acetate esters, and alcohols
   of plant and microbial origin, have been shown to strongly inhibit
   toxigenic fungus growth and aflatoxin formation
   [[46]7,[47]8,[48]9,[49]10]. Fumigation with natural volatiles is an
   ideal method in controlling A. flavus, as it ensures that food is
   protected from pathogenic fungi with reduced or no organoleptic changes
   [[50]11]. Furthermore, volatiles are easily volatilized at ambient
   temperature. This characteristic gives volatile compounds a great
   advantage from the point of view of application practicality and
   homogeneity [[51]12]. Among these volatiles, ethers, such as dimethyl
   disulfide and dimethyl sulfide, have been proved to be effective agents
   for combating pathogens [[52]13,[53]14]. Previously, we demonstrated
   that benzenamine has great capacity for controlling the growth of A.
   flavus [[54]15]. However, the inhibitory effects have not yet been
   studied in depth. It is not clear whether aflatoxin production is
   affected, and the underlying mechanisms are not known.

   The genomes of several species of Aspergillus have recently been
   sequenced and analyzed, and the regulation of aflatoxin biosynthesis
   and development in A. flavus has been well studied [[55]16]. The
   biosynthetic pathway of aflatoxins has been essentially clarified
   [[56]17]. In addition, the functions of several global regulatory
   genes, such as laeA and veA, which are involved in fungal secondary
   metabolism and development, have been characterized [[57]18,[58]19].
   High-throughput sequencing technologies are currently revolutionizing
   the field of biology and RNA sequencing (RNA-Seq) has been applied to
   study a range of eukaryotic transcriptomes, with less sampling bias,
   higher resolution, and much broader expression range coverage
   [[59]20,[60]21].

   In this study, we are interested in revealing the antimicrobial
   activity of benzenamine against A. flavus. The RNA-Seq approach was
   applied to systematically investigate the mechanism of
   benzenamine-induced regulation of the development, aflatoxin
   biosynthesis, and virulence of A. flavus. This work will be meaningful
   for further understanding the interactions of volatiles with A. flavus
   and the regulation of aflatoxin biosynthesis, and the results should be
   of interest to those that are studying the management of A. flavus
   contamination in agricultural products.

2. Results and Discussion

2.1. Antagonistic Activity of Benzenamine against A. flavus

   Fungal colony diameter, aflatoxin production, and colonization of maize
   were quantified to define the inhibitory effect of benzenamine in the
   development, toxigenicity, and virulence of A. flavus. As shown in
   [61]Figure 1, benzenamine exerted inhibitory effects on the mycelial
   growth and spore germination of A. flavus at the tested concentrations.
   Increasing concentrations of benzenamine (from 25 to 400 μL/L) resulted
   in a significant increase in growth inhibition (from 9.67 to 100%).
   Untreated mycelia grew to a diameter of 4.60 cm by three days
   post-inoculation, and conidia germinated completely within 9 h. The
   inhibition of hyphal growth and conidial germination of A. flavus
   resulting from treatment with 100 µL/L of benzenamine was 52.19% and
   73.96%, respectively. Additionally, the minimum inhibitory
   concentration (MIC) of benzenamine against A. flavus was found to be
   200 μL/L. The growth and conidial germination of A. flavus were
   completely inhibited at this concentration. Interestingly, we noted
   that exposing A. flavus to benzenamine for three days inhibited the
   fungus, but it renewed its growth after being transferred into fresh
   Potato Dextrose Agar (PDA) plates. This phenomenon clearly indicates
   that benzenamine suppressed A. flavus growth but did not kill A.
   flavus.

Figure 1.

   [62]Figure 1
   [63]Open in a new tab

   Effects of benzenamine on the hyphal growth and spore germination of
   Aspergillus flavus. The applied concentrations of benzenamine were 25,
   50, 100, 200, and 400 µL/L. Results are presented as the mean ± SD.

   Subsequently, 100 μL/L of benzenamine with moderate bioactivity was
   applied to further investigate the inhibitory effect of benzenamine on
   the toxigenicity and virulence of A. flavus. [64]Figure 2 shows the
   effect of benzamine treatment on aflatoxin B1 production. The
   concentration of aflatoxin B1 was 83.14 ng/g in control groups (CG),
   whereas no aflatoxin B1 (<0.03 ng/g) was detected in A. flavus that was
   treated with benzenamine (EG—experimental group). The results for maize
   that was colonized by A. flavus are shown in [65]Figure 3. In untreated
   maize kernels (CG), inoculation with A. flavus caused the complete
   colonization (3.28 × 10^6 conidia/mL) within five days. In the
   treatments exposing infected kernels to 100 μL/L of benzenamine (EG),
   no visible symptoms were observed, and the number of conidia sharply
   decreased to 0.25 × 10^6 conidia/mL. The results of the antifungal
   ability experiment clearly indicate that benzenamine displays strong
   inhibitory effects on the development, aflatoxin biosynthesis, and
   fungal virulence of A. flavus.

Figure 2.

   [66]Figure 2
   [67]Open in a new tab

   Effects of 100 µL/L of benzenamine on aflatoxin production. (A)
   Morphological characterization of Aspergillus flavus in the absence
   (CG) and presence (EG) of benzenamine. (B) Aflatoxin B1 accumulation by
   Aspergillus flavus in the absence (CG) and presence (EG) of
   benzenamine. The results are presented as mean ± SD. Asterisks indicate
   a significant difference between groups (*** p < 0.001), N. D. denotes
   not detected (<0.03 ng/g).

Figure 3.

   [68]Figure 3
   [69]Open in a new tab

   Effects of 100 µL/L of benzenamine on Aspergillus flavus infection in
   maize. (A) CG: maize inoculated with Aspergillus flavus at five days
   post-inoculation; EG: maize inoculated with Aspergillus flavus exposed
   to benzenamine for five days. (B) The production of Aspergillus flavus
   conidia on maize in CG and EG. Results are presented as the mean ± SD.
   Asterisks indicate a significant difference between groups (*** p <
   0.001).

2.2. Transcriptome Overview

   To identify A. flavus genes that were differentially regulated during
   continuous exposure to benzenamine, a transcriptome analysis of A.
   flavus with three biological replicates was performed using the
   Illumina platform. Raw sequencing data can have issues regarding low
   quality, which can significantly distort analytical results and lead to
   erroneous conclusions. Therefore, quality control steps were performed
   to ensure that RNA-Seq data were of high quality. The clean reads were
   obtained by trimming the raw data containing adapters, poor-quality
   bases (<Q20), and a sequence length smaller than 50 nucleotides
   ([70]Table S1). After the assembly of clean data, a total of 23,639
   unigenes were obtained, with a mean length of 1437 bp ([71]Table S2).

2.3. Annotation and Analysis of All Unigenes

   To understand the transcriptome of A. flavus, all of the unigenes were
   aligned against several databases using BLASTx (E-value ≤ 10^–5),
   including NR (NCBI non-redundant protein sequences), GO (Gene
   Ontology), KEGG (Kyoto Encyclopedia of Genes and Genome), eggNOG
   (evolutionary genealogy of genes: Non-supervised Orthologous Groups),
   and Swiss-Prot. The results are summarized in [72]Table S3. A total of
   14,684 unigenes were matched to known proteins in the NR database,
   although the genome of A. flavus is estimated to contain 13,485 genes
   [[73]22]. This may be due to the variety in the transcripts from
   processes, such as alternative splicing and posttranscriptional
   regulation [[74]23]. The results indicate that more unigenes have the
   potential for translation into functional proteins, which serves to
   improve the annotation of the A. flavus genome.

2.4. Functional and Pathway Enrichment Analysis of Differentially Expressed
Genes

   Out of the 23,639 unigenes, 3589 showed differential accumulation of
   mRNAs. Among all differentially expressed genes (DEGs), 1890 genes
   (accounting for 52.66% of all DEGs) were significantly downregulated
   and 1699 genes (accounting for 47.34% of all DEGs) were upregulated
   compared with the untreated samples ([75]Figure 4, [76]Table S4). All
   DEGs were subjected to GO and KEGG enrichment analyses. A total of 2539
   DEGs were mapped to 2836 GO terms. Among these, 1773, 653, and 410 GO
   terms belong to the biological process, molecular function, and
   cellular component categories, respectively. As shown in [77]Figure 5A,
   transition metal ion binding, metal ion binding, zinc ion binding,
   cation binding, and DNA binding are significant enrichment terms that
   belong to the molecular function category. The significant functional
   terms in the cellular component category are related to the nucleus,
   the intrinsic/integral component of the membrane, and intracellular
   membrane-bounded organelles. Additionally, ncRNA processing and the
   nucleic acid metabolic process are the most abundant in the biological
   process category. According to the KEGG pathway database, significantly
   enriched pathways include the biosynthesis and metabolism of amino
   acids (tyrosine metabolism; phenylalanine metabolism; alanine,
   aspartate, and glutamate metabolism; etc.), purine metabolism, protein
   processing in the endoplasmic reticulum, and mismatch repair, amongst
   others ([78]Figure 5B). Thus, the RNA-Seq results indicate that
   benzenamine exerts complex regulatory effects on A. flavus. Next, the
   genes that are involved in the development, aflatoxin biosynthesis, and
   virulence of A. flavus were further analyzed.

Figure 4.

   [79]Figure 4
   [80]Open in a new tab

   Number of genes showed up-regulated and down-regulated expression in
   experimental group (EG) vs. control group (CG).

Figure 5.

   [81]Figure 5

   [82]Figure 5
   [83]Open in a new tab

   Gene Ontology (GO) functional classification and Kyoto Encyclopedia of
   Genes and Genome (KEGG) pathway enrichment of differentially expressed
   genes when Aspergillus flavus was treated with benzenamine. (A)
   Functional categories at three developmental stages based on GO
   enrichment analysis of differentially expressed genes (DEGs), and the
   asterisk means significant enrichment (* corrected p-value <0.05). (B)
   KEGG pathway enrichment analysis of DEGs.

2.5. Analysis of DEGs Involved in Development

   To elucidate the effects of benzenamine on the development of A.
   flavus, DEGs that are related to the cell wall, cell membrane, conidia,
   transcription factors, and others were analyzed in our work ([84]Table
   1).

Table 1.

   Transcript abundance of genes involved in Aspergillus flavus
   development.
   Gene ID Gene Name Log2
   (EG/CG) * p-Value Function
   Cell wall
   TRINITY_DN6715_c0_g1 crh11 −3.98 8.95 × 10^−8 cell wall glucanase
   TRINITY_DN10780_c1_g3 ags1 −2.24 1.53 × 10^−5 Alpha-1,3-glucan synthase
   TRINITY_DN17727_c0_g1 fks1 −1.38 8.42 × 10^−3 beta-1,3 -glucan synthase
   TRINITY_DN9747_c0_g1 rho2 1.42 2.71 × 10^−4 GTP-binding protein
   TRINITY_DN9087_c0_g1 rho4 1.31 5.24 × 10^−3 GTP-binding protein
   TRINITY_DN11443_c2_g2 cfmA −1.51 3.20 × 10^−4 GPI-anchored CFEM domain
   protein A
   TRINITY_DN11909_c4_g1 afuA −1.75 2.47 × 10^−3 GPI-anchored membrane
   protein
   TRINITY_DN9375_c0_g1 chs6 −1.02 1.65 × 10^−3 Chitin synthase
   TRINITY_DN10195_c0_g1 chs8 −1.31 1.13 × 10^−3 Chitin synthase
   Cell membrane
   TRINITY_DN8232_c0_g1 erg3 3.58 1.91 × 10^−2 C-5 sterol desaturase
   TRINITY_DN10457_c0_g1 erg4 −2.87 8.85 × 10^−6 C6 transcription factor
   TRINITY_DN7343_c0_g1 erg5 −1.80 5.19 × 10^−3 Cytochrome P450
   TRINITY_DN6009_c0_g1 erg6 −1.23 4.12 × 10^−2 24-C-methyltransferase
   TRINITY_DN9690_c0_g1 erg7 −6.36 6.64 × 10^−21 Lanosterol synthase
   TRINITY_DN11958_c0_g2 erg13 −1.34 2.89 × 10^−2
   Hydroxymethylglutaryl-CoA synthase
   TRINITY_DN10380_c0_g1 erg25 −1.64 1.37 × 10^−2 Methylsterol
   monooxygenase
   TRINITY_DN15242_c0_g1 erg26 −1.59 1.48 × 10^−3
   Sterol-4-alpha-carboxylate 3-dehydrogenase
   Conidia
   TRINITY_DN11281_c0_g1 brlA −1.27 8.69 × 10^−4 C[2]H[2] type master
   regulator of conidiophore development
   TRINITY_DN11770_c0_g1 wetA −3.99 1.85 × 10^−21 developmental regulatory
   protein
   TRINITY_DN9659_c0_g1 abaA −3.17 1.23 × 10^−12 Conidiophore development
   regulator
   TRINITY_DN11518_c0_g4 rodA −9.10 6.30 × 10^−19 Conidial hydrophobin
   TRINITY_DN15172_c0_g1 rodB −6.94 4.97 × 10^−23 Conidial hydrophobin
   TRINITY_DN11879_c4_g1 stuA −1.54 7.43 × 10^−6 Cell pattern
   formation-associated protein
   Transcription regulator
   TRINITY_DN9345_c0_g1 laeA −1.82 5.63 × 10^−5 Secondary metabolism
   regulator
   TRINITY_DN11910_c4_g1 veA 1.42 2.68 × 10^−3 Developmental and secondary
   metabolism regulator
   TRINITY_DN8021_c0_g1 fig1 −2.59 4.64 × 10^−3 Ca^2+ regulator and
   membrane fusion protein
   TRINITY_DN10388_c0_g1 sebA −2.44 2.36 × 10^−4 C[2]H[2] finger domain
   transcription factor
   TRINITY_DN12068_c7_g5 mtfA 1.06 2.65 × 10^−2 C[2]H[2] finger domain
   transcription factor
   TRINITY_DN12136_c0_g2 hir3 −1.57 1.24 × 10^−3 Histone transcription
   regulator
   Developmental signal
   TRINITY_DN7900_c0_g1 fluG −2.25 3.28 × 10^−5 Extracellular
   developmental signal biosynthesis protein
   Apoptosis
   TRINITY_DN12180_c5_g1 cycA 2.33 5.30 × 10^−5 Cytochrome c
   G-protein
   TRINITY_DN10123_c0_g1 gblP −1.90 1.37 × 10^−6 Nucleotide-binding
   protein subunit beta-like protein
   TRINITY_DN9739_c0_g1 gna12 −2.16 2.48 × 10^−6 G-protein alpha subunit
   [85]Open in a new tab

   (*): CG, control group; EG, experimental group. Log2 (EG/CG) ≥1
   indicate up-regulated expression and Log2 (EG/CG) ≤−1 indicate
   down-regulated expression.

2.5.1. DEGs Involved in the Cell Wall

   The cell wall provides fungi with a protective barrier against
   environmental stresses, and it is essential for the survival of the
   fungus during development and reproduction [[86]24]. It has been
   reported that the cell wall is an important molecular target of
   antifungal compounds [[87]25]. Numerous DEGs that are involved in the
   cell wall were found in this study ([88]Table 1).

   α-1,3-Glucan and β-1,3-Glucan play critical roles in maintaining the
   normal morphology of the fungal cell wall. Ags1 encodes a synthase that
   mediates the synthesis of α-1,3-glucan [[89]26]. The enzyme
   β-1,3-glucan synthase, encoded by fks1, is an essential and unique
   structural component of β-1,3-Glucan [[90]27]. In the current work,
   ags1 and fks1 were significantly downregulated by benzenamine. Chitin
   is an important structural polysaccharide of the cell wall, and chitin
   synthesis is directly governed by chitin synthase [[91]28]. The
   transcription of chitin synthase genes chs6 and chs8 was moderately
   downregulated. Chitinase is conducive to fungal cell separation during
   their reproduction period. The downregulation of the glucanase gene
   crh11 was also found, and this could lead to fungal reproduction
   disorder [[92]29].

   Glycosylphosphatidyl-inositol (GPI)-anchored proteins are one of the
   major cell wall components. GPI-anchored proteins are essential for the
   normal function of glucan assembly [[93]30]. The genes that are
   responsible for GPI-anchored protein biosynthesis are potential targets
   of antifungal reagents. The RNA-Seq data show that cfmA (GPI-anchored
   CFEM domain protein) and afuA (GPI-anchored membrane protein) had
   reduced expression in our study. In addition, the regulatory subunit of
   the rho family of GTPases is essential to the cell wall integrity
   signaling pathway. It has been proved that the deletion of the rho
   protein results in cytoplasmic leakage [[94]25]. Interestingly, the
   GTP-binding proteins rho2 and rho4 had 2.68- and 2.48-fold increases in
   expression, respectively. This phenomenon may be a defensive response
   of cells to overcome external stimulation [[95]31].

2.5.2. DEGs Involved in the Cell Membrane

   The cell membrane plays important roles in the maintenance of osmotic
   pressure and normal physiological function; thus, it is another
   important target of the antimicrobial substance [[96]32]. Ergosterol is
   an important and specific component of the fungal cell membrane, and it
   is essential for fungal growth and development [[97]33]. Ergosterol is
   considered to be crucial in regulating cell membrane fluidity,
   permeability, and membrane-bound enzyme activities, as well as in
   substance transportation [[98]34]. Furthermore, ergosterol can
   stimulate the growth and proliferation of fungi [[99]35]. As shown in
   [100]Table 1, eight ergosterol biosynthesis genes displayed significant
   expression. Of these eight, seven DEGs were downregulated, exhibiting
   fold changes that ranged between 2.35- and 82.03-fold. The erg7 gene
   encodes lanosterol synthase and experienced the biggest reduction.
   However, the erg3 gene was upregulated by 11.64-fold. It was reported
   that the last reactions in ergosterol biosynthesis are catalyzed by
   erg3/erg4/erg5, and these enzymes catalyze the conversion of episterol
   into ergosterol [[101]36]. Therefore, increased expression of erg3 may
   be considered to be a compensation response to the downregulation of
   erg4/erg5.

2.5.3. DEGs Involved in Conidia

   Asexual sporulation is fundamental to the ecology and lifestyle of
   fungi. The ability to produce conidia is a key factor contributing to
   the fecundity, propagation, and fitness of A. flavus. The formation and
   maturation of conidia is primarily governed by the brlA-abaA-wetA
   regulatory cascade [[102]37,[103]38]. In the current study, the brlA,
   abaA, and wetA genes were significantly downregulated to different
   degrees, directly leading to lower levels of conidia formation. In
   addition, the transcription of the hydrophobin genes rodA and rodB was
   remarkably downregulated ([104]Table 1). The formation of rodlets,
   physical resistance, and immunological inertia of the conidia is partly
   due to the presence of a hydrophobic layer that is composed of a
   protein from the hydrophobin family [[105]39]. These identified DEGs
   indicate that benzenamine hinders the normal formation and
   physiological state of A. flavus conidia.

2.5.4. DEGs Involved in Transcription Factors

   Numerous transcription factor-encoding genes that are related to fungal
   development were differentially expressed in A. flavus after exposure
   to benzenamine. Most of the transcription factors, such as the
   secondary metabolism regulator laeA, C[2]H[2] finger domain
   transcription factor sebA, and Ca^2+ regulator and membrane fusion
   protein fig1, were downregulated to varying extents, whereas some
   transcription factors were upregulated, including the C[2]H[2]-like
   transcription factor mtfA and the developmental and secondary
   metabolism regulator veA. Among these, laeA and veA are the most
   important regulatory genes in Aspergillus spp. A complicated network of
   global regulators governs the development and secondary metabolism in
   Aspergillus spp. by including laeA and veA [[106]19]. LaeA was first
   identified in Aspergillus nidulans, and it has been extensively
   studied. Numerous developmental genes are regulated by laeA, such as
   knh1, encoding a GPI-anchored protein involved in cell wall
   biosynthesis; stuA, encoding a cell pattern formation-associated
   protein that is related to conidiophore development; and, hydrophobic
   proteins encoded by rodA/B [[107]40]. VeA also has the ability to
   regulate developmental genes, such as the downregulation of the asexual
   development-associated transcription factors brlA and abaA, which are
   required for the formation of conidia [[108]38,[109]41]. A
   heterotrimeric complex that is formed by the proteins encoded by laeA,
   veA, and velB has been reported to regulate sporulation and secondary
   metabolism [[110]42]. In addition, it was reported that laeA and veA
   negatively affect each other’s transcription [[111]19,[112]43].

   The transcriptomic results reveal that benzenamine exerted different
   effects on the expression of laeA and veA in the current work. The gene
   laeA was significantly downregulated by 3.57-fold, whereas veA was
   upregulated by 2.67-fold. This phenomenon may be due to the opposing
   regulatory effects of laeA and veA. The results indicate that
   benzenamine may suppress the development of A. flavus through its
   adverse effects on key processes, such as cell wall synthesis and
   conidia production, by regulating the expression of laeA and veA. This
   is also consistent with the above analysis results. In addition, the
   gene cytC, which encodes an apoptogenic factor, was significantly
   upregulated (5.03-fold). We hypothesize that cytC may be regulated by
   laeA or veA. However, up to now, regulation by laeA or veA of genes
   that are related to apoptosis has not been described, and we will
   investigate this inference in our future work.

2.6. Analysis of DEGs Involved in Aflatoxin Biosynthesis

2.6.1. DEGs Involved in Aflatoxin Biosynthesis

   To evaluate the regulatory roles of benzenamine on aflatoxin
   biosynthesis, the expression levels of genes that are involved in
   aflatoxin biosynthesis were analyzed. The biosynthetic pathways of
   aflatoxin have been well described [[113]44,[114]45]. A total of 10
   genes that are involved in the aflatoxin biosynthesis, especially aflA,
   aflB, aflD, aflT, and aflU, were downregulated by benzenamine
   ([115]Table 2). Two fatty acid synthases, aflA and aflB, are related to
   the early stage of aflatoxin biosynthesis, and they are capable of
   converting acetate to norsolorinic acid (NOR), which is a stable
   aflatoxin precursor [[116]46,[117]47]. On the other hand, there was no
   significant difference in the expression of aflC, which has an
   equivalent function. The expression level of aflD was reported to play
   a significant role in aflatoxin biosynthesis. The gene aflD encodes a
   norsolorinic acid ketoreductase that converts NOR to averantin (AVN)
   [[118]48]. Additionally, aflE and aflF, homologous to aflD, are
   predicted to catalyze NOR to AVN [[119]47,[120]49]. The expression of
   aflF was downregulated by 3.13-fold, and aflE showed no significant
   difference in expression. AflT encodes a transmembrane protein and it
   is located at the end of the gene cluster for aflatoxin biosynthesis
   [[121]50]. It was reported that aflT is not essential for the
   production and secretion of aflatoxin, and the expression of aflT is
   not regulated by the transcription regulator genes aflR and aflS, but
   by fadA, which encodes a G alpha protein-dependent signaling pathway
   [[122]51]. Furthermore, aflR encodes a specific zinc-finger DNA-binding
   protein, which is an important regulatory gene that is required for
   transcriptional activation of most genes in aflatoxin biosynthesis
   [[123]47,[124]52]. The downregulation of aflR by benzenamine could
   cause changes in other aflatoxin biosynthesis pathway genes. However,
   the expression level of another regulatory gene, aflS, did not display
   any obvious changes. Our data clearly demonstrate that the aflatoxin
   production of A. flavus treated by benzenamine is directly reduced by
   downregulating the expression levels of aflatoxin biosynthesis genes.

Table 2.

   Transcript abundance of genes that are involved in aflatoxin
   biosynthesis.
   Gene ID Gene Name Log2
   (EG/CG) * p-Value Function
   Aflatoxin biosynthesis
   TRINITY_DN9945_c0_g1 aflA −3.42 5.80 × 10^−10 Fatty acid synthase alpha
   subunit
   TRINITY_DN6952_c0_g1 aflB −3.32 4.61 × 10^−2 Fatty acid synthase
   subunit beta
   TRINITY_DN61_c0_g1 aflD −4.96 2.64 × 10^−8 Norsolorinic acid
   ketoreductase
   TRINITY_DN12173_c2_g2 aflF −1.65 4.36 × 10^−2 Norsolorinic acid
   ketoreductase
   TRINITY_DN10698_c0_g2 aflO −1.83 5.63 × 10^−3 O-methyltransferase
   TRINITY_DN10938_c0_g1 aflQ −1.49 2.16 × 10^−4 Oxidoreductase
   TRINITY_DN10167_c0_g1 aflR −1.86 1.68 × 10^−2 Aflatoxin biosynthesis
   regulatory protein
   TRINITY_DN9418_c0_g1 aflT −2.72 4.27 × 10^−2 Transmembrane protein
   TRINITY_DN399_c0_g1 aflU −3.24 2.31 × 10^−8 P450 monooxygenase
   TRINITY_DN10373_c0_g1 aflW −1.60 1.37 × 10^−2 FAD-binding monooxygenase
   Carbon metabolism
   TRINITY_DN7925_c1_g1 creA −1.14 9.62 × 10^−5 DNA-binding protein
   TRINITY_DN10031_c0_g1 mexAM 1.55 1.84 × 10^−2 Oxidoreductase
   TRINITY_DN10394_c0_g1 pot1 1.90 1.45 × 10^−6 Acetyl-CoA
   C-acyltransferase
   TRINITY_DN11110_c0_g1 rntA 1.88 5.21 × 10^−6 Guanyl-specific
   ribonuclease
   TRINITY_DN11497_c0_g1 ppoA −1.83 7.67 × 10^−6 Linoleate 8R-dioxygenase
   like protein
   TRINITY_DN12116_c3_g3 ppoC −2.79 9.25 × 10^−4 Fatty acid oxygenase
   Nitrogen metabolism
   TRINITY_DN5952_c0_g1 nmrAL1 −1.60 2.14 × 10^−4 NmrA-like family
   domain-containing protein 1
   TRINITY_DN11972_c8_g2 gdh-1 −2.42 1.52 × 10^−9 Specific glutamate
   dehydrogenase
   TRINITY_DN11667_c1_g3 glt1 −2.40 4.57 × 10^−5 Putative glutamate
   synthase
   TRINITY_DN11707_c0_g1 niiA −1.73 4.23 × 10^−8 Nitrite reductase
   TRINITY_DN10046_c0_g1 nirA −1.93 2.28 × 10^−4 Nitrogen assimilation
   transcription factor
   TRINITY_DN13321_c0_g1 ddc 1.36 1.02 × 10^−2 Aromatic-L-amino-acid
   decarboxylase
   TRINITY_DN9125_c0_g1 aat2 −3.66 1.66 × 10^−2 Aspartate aminotransferase
   TRINITY_DN19445_c0_g1 melO −3.40 8.11 × 10^−8 Tyrosinase
   TRINITY_DN10664_c0_g1 orsC −1.06 4.78 × 10^−2 Tyrosinase-like protein
   TRINITY_DN11859_c0_g1 gad1 −4.38 1.27 × 10^−27 Glutamate decarboxylase
   TRINITY_DN11916_c1_g2 gfa1 −1.46 2.29 × 10^−4
   Glutamine--fructose-6-phosphate aminotransferase
   TRINITY_DN12169_c4_g1 sch9 1.86 2.72 × 10^−4 Serine/threonine-protein
   kinase
   Purine metabolism
   TRINITY_DN12187_c1_g2 ade17 −1.58 6.35 × 10^−5 Bifunctional purine
   biosynthesis protein
   TRINITY_DN8936_c0_g1 uaY −3.71 2.76 × 10^−4 Positive regulator of
   purine utilization
   TRINITY_DN9209_c0_g1 uapC −3.11 1.58 × 10^−9 Purine permease
   TRINITY_DN9614_c0_g1 fcy2 −2.36 3.47 × 10^−4 Purine-cytosine permease
   TRINITY_DN10376_c0_g2 pol12 −2.28 3.34 × 10^−2 DNA polymerase alpha
   subunit B
   TRINITY_DN8807_c0_g1 hxA −1.16 2.02 × 10^−3 Xanthine dehydrogenase
   cAMP signaling pathway
   TRINITY_DN10852_c0_g2 ATP12A −2.10 8.03 × 10^−3 ATPase alpha 1 subunit
   TRINITY_DN11913_c3_g2 pld1 −1.55 8.45 × 10^−5 Phospholipase D1
   TRINITY_DN11065_c0_g2 pka-C3 −1.02 1.11 × 10^−2 cAMP-dependent protein
   kinase
   [125]Open in a new tab

   (*): CG, control group; EG, experimental group. Log2 (EG/CG) ≥ 1
   indicate up-regulated expression and Log2 (EG/CG) ≤ −1 indicate
   down-regulated expression.

2.6.2. DEGs Involved in Carbon/Nitrogen Metabolism

   It has been reported that aflatoxin production is influenced by
   nutrition factors, such as carbon and nitrogen sources
   [[126]53,[127]54]. Several DEGs that are involved in carbon and
   nitrogen metabolism were found in our data. Carbon catabolite
   repression (CCR) is a regulatory phenomenon that is hierarchically
   implemented to organize carbohydrate utilization, which is required for
   the regulation of growth and secondary metabolism in fungi
   [[128]55,[129]56]. CreA, which is a global regulator of CCR, encodes a
   zinc finger of a Cys2/His2 class protein and mediates various
   alternative carbon-utilizing systems [[130]57,[131]58]. The deletion of
   creA induces a strong reduction of aflatoxin synthesis. Additionally,
   cell wall homeostasis and conidial differentiation are regulated by
   creA [[132]54,[133]59]. The expression levels of the creA transcript in
   A. flavus after exposure to benzenamine were significantly lower than
   in the control. It is supposed that creA regulates gene expression by
   binding to consensus binding sites in the promoters of target genes,
   and this consensus binding site has been found in most aflatoxin gene
   promoter regions [[134]60,[135]61]. It appears likely that the
   downregulation of creA by benzenamine also contributes to the
   depression of aflatoxin production.

   Nitrogen source is another important nutritional factor that is linked
   with aflatoxin biosynthesis [[136]62,[137]63]. Microorganisms can use a
   wide range of nitrogen sources, and different nitrogen sources may have
   different effects on aflatoxin production [[138]53]. For example, it
   has been reported that glutamine and tyrosine favor aflatoxin
   production in A. flavus, while tryptophan does not [[139]64,[140]65].
   Nitrogen utilization is often mediated by nitrogen metabolite
   repression (NMR) [[141]66]. The gene nmrA negatively regulates several
   genes that are involved in NMR and it appears to be involved in the
   development and aflatoxin biosynthesis in A. flavus. Furthermore, the
   absence of nmrA results in reduced aflatoxin production [[142]67]. The
   results of RNA-Seq in our study show that the expression of nmrAL1,
   which encodes nmrA-like family domain-containing protein 1, was
   significantly decreased. Additionally, gad1 and gfa1, which are
   involved in the glutamine metabolic process, were downregulated by
   benzenamine.

2.6.3. Other Related DEGs

   Previous reports have demonstrated that pathway-specific regulators, as
   well as a complicated network of global regulators, govern multiple
   secondary metabolite gene clusters [[143]19,[144]43]. The expression of
   aflatoxin biosynthesis cluster genes is also modified by the global
   regulator laeA. The deletion of laeA blocks the production of aflatoxin
   by downregulating the expression of early aflatoxin biosynthesis genes
   and the pathway-specific transcriptional regulator aflR [[145]18]. In
   addition, conidial development and aflatoxin formation are tightly
   coordinated in A. flavus [[146]43]. The loss of conidial hydrophobicity
   in the laeA deletion mutant is considered to be capable of influencing
   the formation and stability of vesicles, thereby reducing aflatoxin
   biosynthesis [[147]18,[148]68]. Additionally, the loss of laeA may
   downregulate the expression of nmrA, and this regulation of nmrA
   contributes to reduced aflatoxin biosynthesis [[149]67]. Our data agree
   with those indicating comprehensive regulation in A. flavus by laeA.

   The cAMP/PKA signaling pathway regulates fungal morphogenesis and
   metabolism, including mycotoxin biosynthesis
   [[150]69,[151]70,[152]71,[153]72]. Previous papers have shown that cAMP
   signaling plays an important role in hyphal growth, conidiation, and
   production of DON [[154]73,[155]74]. It was reported that decreasing
   levels of cAMP block aflatoxin biosynthesis in A. flavus
   [[156]75,[157]76]. In the current work, all three DEGs in the cAMP
   signaling pathway were downregulated by magnitudes that ranged from
   2.02- to 4.30-fold. The results indicate that the downregulation of the
   cAMP pathway genes by benzenamine is likely to negatively regulate
   aflatoxin biosynthesis in A. flavus. Additionally, of interest, most of
   the genes that are involved in purine metabolism were significantly
   downregulated by benzenamine, which implies that this process might
   have a role in aflatoxin synthesis.

   The RNA-Seq data indicate that benzenamine not only directly reduces
   the production of aflatoxin by downregulating the aflatoxin
   biosynthesis pathway genes and pathway-specific regulatory genes, but
   it also indirectly blocks aflatoxin synthesis by mediating nutrient
   metabolism, signaling pathways, and the expression of the global
   transcription regulator.

2.7. Analysis of DEGs Involved in the Virulence of A. flavus

2.7.1. DEGs Involved in Hydrolases

   Extracellular hydrolases, such as glucosidase, proteases, and lipases,
   are critical for A. flavus to colonize its hosts
   [[158]49,[159]77,[160]78]. A. flavus is able to degrade complex organic
   substrates, obtain nutrients for growth, macerate, and then invade host
   tissues [[161]79]. The decreased abundance of hydrolases increases the
   difficulty for mycelia to penetrate and colonize hosts [[162]40].
   Several A. flavus hydrolytic enzymes, including α-glucosidase, lipase,
   and neutral protease, were downregulated to different degrees,
   according to our results ([163]Table 3). Additionally, cutinase
   transcription factors ctf1A/B showed significantly downregulated
   transcription. However, of interest, there was no significant
   difference in the expression of cutinase in A. flavus after exposure to
   benzenamine.

Table 3.

   Transcript abundance of genes that are involved in virulence of
   Aspergillus flavus.
   Gene ID Gene Name Log2
   (EG/CG) * p-Value Function
   Hydrolase
   TRINITY_DN11687_c2_g1 mal1 −3.39 6.39 × 10^−10 Alpha-glucosidase
   TRINITY_DN18434_c0_g1 mal2 −2.86 2.02 × 10^−3 Alpha-glucosidase
   TRINITY_DN9284_c0_g1 agdC −2.16 5.88 × 10^−6 Alpha -glucosidase
   TRINITY_DN4002_c0_g1 lip −3.41 2.15 × 10^−3 Lipase
   TRINITY_DN11096_c0_g3 NPII −7.81 2.84 × 10^−61 Neutral protease
   TRINITY_DN10289_c0_g1 ctf1B −2.77 2.89 × 10^−2 Cutinase transcription
   factor 1 beta
   TRINITY_DN11791_c0_g4 ctf1A −1.59 4.29 × 10^−2 Cutinase transcription
   factor 1 alpha
   TRINITY_DN8062_c0_g1 abfA −2.71 2.69 × 10^−3
   Alpha-L-arabinofuranosidase A
   TRINITY_DN12177_c2_g1 dvrA 1.03 1.97 × 10^−2 C[2]H[2] finger domain
   transcription factor
   [164]Open in a new tab

   (*): CG, control group; EG, experimental group. Log2 (EG/CG) ≥1
   indicate up-regulated expression and Log2 (EG/CG) ≤−1 indicate
   down-regulated expression.

2.7.2. DEGs Involved in the Development and Metabolism of A. flavus

   The virulence of A. flavus has been proved to be multifactorial, and it
   is also tightly coordinated with development, sporulation, and
   metabolism [[165]80]. The cell wall is critical for the virulence of
   fungal pathogenicity [[166]25]. Cell wall components, such as
   polysaccharides and proteins, are considered to be virulence factors
   and they contribute to colonization of the host [[167]81,[168]82]. The
   loss of cell wall integrity might influence the colonization by A.
   flavus of the host [[169]75]. Several genes that are involved in the
   cell wall were downregulated in our study. Therefore, benzenamine might
   reduce the virulence of A. flavus by damaging its cell wall integrity.

   Aspergillus species have the ability to produce a large quantity of
   asexual spores that spread through conidia [[170]83]. Conidia are
   abundantly suspended in the air and environment, and they can remain
   viable for a long period of time [[171]84,[172]85]. These conidia will
   form a short germ tube and germinate when they colonize in hosts
   [[173]86]. The formation and germination of conidia is critical for
   successful colonization. The expression of stuA, which encodes a cell
   pattern formation-associated protein that is involved in conidiophore
   development, was downregulated by benzenamine. Additionally, in
   Aspergillus, conidial hydrophobicity plays an important role in the
   infection of host tissues. The insoluble hydrophobic rodlet layer that
   is enveloped in the surface of A. flavus conidia contributes to the
   strengthening of the dispersal capacity and survival in a hostile
   environment, and the rodlet layer comprises the hydrophobic rodA
   protein that covalently binds to the conidial cell wall via GPI
   remnants [[174]39,[175]87]. Previous studies have reported that
   decreased conidial hydrophobicity accompanies reduced pathogenicity
   [[176]88]. Our data demonstrate that hydrophobins rodA and rodB were
   also significantly decreased in abundance. The results indicate that
   conidial hydrophobins may be possible targets for preventing A. flavus
   infection in maize, which is in agreement with the above statement.

   Carbon and nitrogen nutrients are required for the growth and secondary
   metabolism of fungi, and the effect of these nutrients on the virulence
   of A. flavus was reported recently. CreA and nmrA play important roles
   in the invasive virulence of A. flavus. The deletion of creA causes a
   defect in its capacity to effectively infect the host due to a
   reduction in conidial quantity and hydrophobicity [[177]54]. Similarly,
   the loss of nmrA decreases the virulence of A. flavus, compromising its
   ability to produce conidia and colonize the host [[178]67]. Thus,
   according to our data, benzenamine might reduce the pathogenicity of A.
   flavus by decreasing the expression of creA and nmrA.

   In addition, the global regulator laeA and cAMP signaling have been
   reported to regulate the virulence of A. flavus. Previous studies
   reported that the deletion of laeA decreases the ability to colonize
   seeds [[179]40,[180]43]. In laeA mutant strains, the expression of
   several genes that are vital for pathogenicity (such as lipase,
   α-amylase, nmrA, and rodA) is downregulated, which might result in
   reduced fungal virulence [[181]18,[182]67]. The involvement of the cAMP
   signaling pathway in the regulation of fungal virulence has been
   reported [[183]89,[184]90]. The loss of genes in the cAMP signaling
   pathway, such as acyA and cpk1, considerably reduces the virulence of
   pathogenic fungi by repressing conidial production
   [[185]74,[186]76,[187]91]. In the present study, our data demonstrate
   that benzenamine could decrease the virulence of pathogenic A. flavus
   by regulating laeA transcription and the cAMP signaling pathway.

2.8. Validation of RNA-Seq Data by qRT-PCR

   The qRT-PCR experiment was used to validate the RNA-Seq data in our
   study. DEGs that are involved in aflatoxin synthesis and the important
   global regulatory factor laeA were chosen for qRT-PCR validation. The
   differential gene expression profiles between the control group (CG)
   and experimental group (EG) are shown in [188]Figure 6. The results
   show that these genes have expression patterns that are consistent with
   the RNA-Seq data, indicating the reliability of the transcriptome
   analysis in the current work. Overall, in order to elucidate the
   regulatory molecular events following A. flavus exposure to
   benzenamine, a hypothetical molecular mode of action is proposed on the
   basis of our data ([189]Figure 7).

Figure 6.

   [190]Figure 6
   [191]Open in a new tab

   Quantitative real-time PCR validation of aflatoxin biosynthesis genes
   and laeA after treatment with benzenamine. CG, control group; EG,
   experimental group. Results are presented as mean ± SD. Log2 (EG/CG) ≤
   −1 indicates downregulated expression.

Figure 7.

   [192]Figure 7
   [193]Open in a new tab

   A schematic diagram for the proposed mechanism of benzenamine against
   Aspergillus flavus.

3. Conclusions

   The current work demonstrates that benzenamine exerts strong inhibitory
   effects on the development, aflatoxin production, and pathogenicity of
   A. flavus, and the results provide fundamental information in
   understanding the fungal response to benzenamine at the transcriptional
   level. Based on the transcriptional profile, thousands of genes are
   downregulated in A. flavus after treatment with benzenamine, including
   genes that are associated with growth, differentiation, and aflatoxin
   biosynthesis in A. flavus, and we conclude that benzenamine inactivates
   A. flavus by suppressing the expression of related genes by
   downregulating the regulatory factor laeA. These enriched DEGs could be
   exploited in order to develop genetic strategies to reduce
   contamination by pathogenic A. flavus.

4. Materials and Methods

4.1. Microorganisms

   A. flavus used in this study was obtained from the China Center of
   Industrial Culture Collection (CICC NO. 2219). The strain was cultured
   on PDA (200 g/L potato infusion, 20 g/L dextrose, and 20 g/L agar) in
   Petri Dishes at 28 °C for three days. Conidia were washed with sterile
   distilled water containing 0.05% (v/v) Tween 80, and their density was
   adjusted by a hemocytometer to a final concentration of approximately 1
   × 10^6 conidia/mL.

4.2. Antifungal Assays

4.2.1. Inhibition of Hyphal Growth of A. flavus

   To study the efficacy of benzenamine against mycelial growth, a setup
   of two inverse face-to-face Petri Dishes was applied according to Wu et
   al. [[194]14]. The system consists of two 6 cm lidless Petri Dishes.
   The upper plate contained 5 mL of PDA inoculated with a 6 mm diameter
   A. flavus agar plug in the center, and the lower plate contained
   benzenamine at different concentrations (25, 50, 100, 200, 400 µL/L).
   The two dishes were sealed with a double layer of parafilm and then
   incubated at 28 °C for three days. The treatments consisted of three
   replicates, and each experiment was performed in triplicate. The
   inhibition rate of mycelial growth was calculated using the following
   Equation:
   Inhibition rate (%) = (Rc − Rt)/Rc × 100% (1)

   where Rc is the colony diameter of the control and Rt is the colony
   diameter of the treatment.

4.2.2. Inhibition of Conidial Germination of A. flavus

   In this assay, face-to-face Petri Dishes, as described above, were
   applied. The top dishes, which contained a sterile filter-paper dipped
   in benzenamine, were attached face-to-face to a PDA plate spread with a
   100 μL conidial suspension of A. flavus. The setup was then incubated
   at 28 °C for 9 h. Conidial germination was evaluated by examining no
   less than 100 conidia per Petri Dish. Conidia were considered to be
   germinated when the germ tube measured at least twice its length
   [[195]92]. The treatments consisted of three replicates, and each
   experiment was performed in triplicate. Inhibition of germination was
   calculated according to the formula:
   Inhibition of germination (%) = (Gc − Gt)/Gc × 100% (2)

   where Gc is the germination rate of the control and Gt is the
   germination rate of the fungus exposed to benzenamine.

4.3. Determination of Aflatoxin B1

   Aflatoxin B1 content was detected with an ELISA kit in this study. A.
   flavus was treated with 100 µL/L of benzenamine for five days at 28 °C.
   Aflatoxin B1 extraction was carried out following the procedure that
   was described by Bavaro et al. [[196]93]. Briefly, five fungal agar
   plugs (6 mm diameter) were extracted with 25 mL of 80% methanol. The
   extraction solution was centrifuged at 4500 rpm for 10 min and the
   supernatant was measured using an ELISA kit (Huaan Magnech Bio-Tech
   Co., Ltd., Beijing, China), according to the manufacturer’s
   instructions. The linear range was between 0.03–2 ng/g for AFB1 (R^2 =
   0.9983). The limit of detection (LOD) was 0.03 ng/g and the limit of
   quantification (LOQ) was 0.08 ng/g. The average spiked recovery rate
   was 100 ± 20%, with the coefficient of variation (CV) being less than
   10%.

4.4. Virulence of A. flavus in Maize

   Undamaged maize kernels were surface sanitized with 0.1% hypochlorite
   and then rinsed with sterile water three times. The maize kernels
   inoculated with the conidial suspension (1 × 10^6 conidia/mL) of A.
   flavus were placed in a lidless plate, and then 100 μL/L of benzenamine
   was distributed evenly on the inner surface of the plate in the form of
   small droplets (0.5 μL). The two dishes were sealed and incubated at 28
   °C for five days, after which the fungal colonization of maize kernels
   was observed on each plate and the colonized kernels were harvested in
   100 mL conical flasks with 20 mL of sterile 0.05% Tween 80 water
   solution. Spores were counted by a hemocytometer.

4.5. Preparation of cDNA Libraries and Illumina Sequencing

   The conidial suspension of A. flavus was spread on PDA medium that was
   overlaid with sterile cellophane and then sequentially exposed to 100
   µL/L of benzenamine for five days at 28 °C. The cellophane membrane
   with mycelia from the whole plate was scraped off with a knife and then
   immediately frozen in liquid nitrogen for RNA extraction. Total RNA was
   isolated using Trizol Reagent (Invitrogen Life Technologies, Carlsbad,
   CA, USA), according to the manufacturer’s instructions. The
   concentration and quality of RNA for each sample were determined by a
   NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE,
   USA), and the integrity of RNA was checked using an Agilent 2100
   Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA).

   The mRNA was enriched from total RNA using poly-T oligo-attached
   magnetic beads and then cleaved into short fragments using divalent
   cations under an elevated temperature in an Illumina proprietary
   fragmentation buffer. The cleaved mRNA fragments were copied into
   first-strand cDNA using random oligonucleotides and Super Script II,
   followed by second-strand cDNA synthesis using DNA Polymerase I and
   RNase H. The remaining overhangs were converted into blunt ends via
   exonuclease/polymerase activities, and the enzymes were then removed.
   After adenylation of the 3′ ends of the DNA fragments, Illumina PE
   adapter oligonucleotides were ligated to prepare for hybridization. The
   adaptor-modified fragments were purified with the AMPure XP system
   (Beckman Coulter, Beverly, CA, USA) in order to preferentially select
   200 bp in length. DNA fragments with ligated adaptor molecules on both
   ends were enriched using the Illumina PCR Primer Cocktail in a 15-cycle
   PCR reaction. The products were purified with the AMPure XP system and
   then quantified using the Agilent high-sensitivity DNA assay on a
   Bioanalyzer 2100 system (Agilent Technologies, Palo Alto, CA, USA). The
   sequencing library was then sequenced with the Hiseq 2500 platform
   (Illumina, San Diego, CA, USA) by Shanghai Personal Biotechnology Cp.
   Ltd.

4.6. De Novo Transcriptome Assembly and Annotation

   De novo transcriptome analysis was performed according to Diao et al.
   [[197]94]. Briefly, the quality control analysis on raw data was done
   by using FastQC (Version 0.11.7, Babraham bioinformatics, Cambridge,
   UK, 2018). Subsequently, the clean reads were obtained by removing
   adaptors and low-quality reads with Cutadapt [[198]95]. The clean reads
   were then assembled de novo using the Trinity platform
   ([199]http://trinityrnaseq.sourceforge.net/). The longest transcripts
   of each gene were regarded as unigenes. All of the unigenes were
   assigned to five databases using BLASTx (E-value ≤ 10^−5), including NR
   (NCBI non-redundant protein sequences), GO (Gene Ontology), KEGG (Kyoto
   Encyclopedia of Genes and Genome), eggNOG (evolutionary genealogy of
   genes: Non-supervised Orthologous Groups), and Swiss-Prot.

4.7. Identification and Analysis of DEGs

   The levels of gene expression were estimated as normalized FPKM
   (fragments per kilobase of transcript per million mapped reads) using
   RSEM (RNA-seq by expectation-maximization) [[200]96]. Differential
   expression analysis was performed with DESeq (Version 1.20.0,
   Bioconductor, New York, NY, USA, 2018) and genes with a fold change >2
   and corrected p-value < 0.05 were set as the threshold for
   significantly differential expression. Finally, GO functional
   annotation and KEGG pathway enrichment analysis were performed to
   uncover the functions of DEGs.

4.8. qRT-PCR Analysis

   To validate the reliability of A. flavus gene expression data obtained
   by RNA-Seq, qRT-PCR was conducted for 10 genes that were involved in
   aflatoxin biosynthesis and the global regulatory gene laeA. The primers
   are listed in [201]Table S5; the β-tubulin gene was selected as the
   endogenous reference gene. Total RNA extraction was performed as
   described above, and cDNA was synthesized with a Takara RNA PCR Kit
   (Takara, Dalian, China). qRT-PCR was performed using an Mx3000p
   instrument (Stratagene, La Jolla, CA, USA) with a final volume of 20 μL
   containing 10 μL of SYBR premix ExTaq, 0.5 μL of each forward and
   reverse primer (10 mM), 2 μL of cDNA template, 0.4 μL of ROX Reference
   Dye, and 6.6 μL of RNase-free water. The comparative 2^−ΔΔCT method was
   employed to calculate relative gene expression [[202]97].

4.9. Availability of Supporting Data

   The raw data that was generated in this study has been deposited in the
   NCBI’s Sequence Read Archive (SRA) database with the accession number
   SRP181717 (BioProject ID: PRJNA516725).

4.10. Statistical Analyses

   Statistical analyses were performed with SPSS for Windows version 20.0
   (SPSS Inc., Chicago, IL, USA, 2011). The data were evaluated by
   Student’s t-test or one-way ANOVA followed by LSD according to the
   experimental design. p < 0.05 was considered to be statistically
   significant.

Supplementary Materials

   The following are available online at
   [203]http://www.mdpi.com/2072-6651/11/2/70/s1, Table S1: Statistics on
   filtering of RNA-Seq Data, Table S2: Summary of transcripts and
   unigenes in this study, Table S3: Summary of annotation results, Table
   S4: Summary of differentially expressed genes, Table S5: List of
   primers used in this study.
   [204]Click here for additional data file.^ (290.2KB, zip)

Author Contributions

   Conceptualization, C.W., L.L. and M.Y.; Performing the experiments,
   M.Y., S.L. and J.Z.; Data processing, M.Y. and H.L.; Writing and
   approving the manuscript, M.Y., Q.G., Z.L., S.W. and C.W.

Funding

   This research was funded by the Key Technologies R & D Program of
   Tianjin [Grant number 16YFZCNC00700]; National Natural Science
   Foundation of China [Grant number 31701668]; Natural Science Foundation
   of Tianjin City [Grant number 17JCQNJC14300].

Conflicts of Interest

   The authors declare no conflict of interest.

Key Contribution

   Fumigation with benzenamine has significant inhibitory effects on the
   development, aflatoxin biosynthesis, and virulence of Aspergillus
   flavus by regulating the expression of a series of genes. This study
   demonstrates that benzenamine has the potential for development as a
   commercial antimicrobial fumigant product, and the RNA-Seq data
   resulting from this study can facilitate finding new potential targets
   for controlling A. flavus.

References