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Differential regulation of mycelial growth and aflatoxin biosynthesis by Aspergillus flavus under different temperatures as revealed by strand‐specific RNA‐Seq

Abstract Although several regulatory pathways have been reported for Aspergillus flavus, the regulation of aflatoxin production and mycelial growth under different temperatures remains unclear. In this study, A. flavus differentially expressed genes (DEGs) and regulatory pathways were analyzed under three temperatures, by strand‐specific RNA‐Seq. Results show that a total of 2,428 and 1,474 DEGs were identified in fungal mycelia cultured at 20°C and 37°C, respectively, as compared with the control (28°C). Approximately ~ 79% of DEGs in the 37°C samples were up‐regulated genes, while ~ 63% of DEGs in the 20°C samples were down‐regulated genes. Most of the DEG pathways enriched by lower temperatures differed from those enriched by higher temperatures, while only a small portion of the pathways were shared by A. flavus grown under different temperatures. Aflatoxin biosynthesis, Butanoate metabolism, oxidation–reduction process, and benzene‐containing compound metabolic process were the shared down‐regulated pathways, while steroid biosynthesis, oxidoreductase activity, cellular protein modification process, DNA binding, protein complex were the shared up‐regulated pathways between lower and higher temperatures. The shared genes and pathways are the key regulatory candidates for aflatoxin biosynthesis with changes of temperature. In addition, the identification of both up‐regulated and down‐regulated genes provides a useful gene set for further investigation of the aflatoxin biosynthesis among Aspergillus.

exposed to aflatoxins via the ingestion of contaminated food or products. Several studies have shown that the consumption of food contaminated with aflatoxins can be harmful to the liver, kidney, epididymis, testis, heart, brain, nervous system and can lead to immune suppression and carcinogenic effects, or even death (Kumar, Mahato, Kamle, Mohanta, & Kang, 2016;Nierman et al., 2015). Although a large number of techniques including physical, chemical, and biological methods have been developed to prevent or reduce the occurrence of aflatoxins in foods, these strategies are not always effective in eliminating grain contamination (Gressel & Polturak, 2018;Klich, 2007;Kumar et al., 2016). To date, the regulatory mechanisms for mycelial growth and aflatoxin biosynthesis under different environmental conditions remain unclear. Studies have also shown that the occurrence of aflatoxins in crops is influenced by various environmental factors, such as nitrogen levels, light, temperature, water, redox status, and pH level (Georgianna & Payne, 2009;Khlangwiset, Shephard, & Wu, 2011). Of these factors, high temperature and drought stress are commonly reported precursors to aflatoxin outbreaks in corn and other crops (Klich, 2007).
Previous investigations have shown that the genes for aflatoxin biosynthesis are clustered in the genome of Aspergillus (Sarma, Bhetaria, Devi, & Varma, 2017;Yu et al., 2004). Twenty-five genes were identified from the 70 kb gene cluster in A. flavus and 82 kb gene cluster in A. parasiticus (Alkhayyat & Yu, 2014;Yu et al., 2004).
Of these, the functions of 19 genes have been assigned, while the structures of at least 15 intermediate gene products have been defined (Alkhayyat & Yu, 2014;Yu et al., 2004). Enzymes convert the initial substrate acetate into the four major aflatoxins (B1, B2, G1, and G2) (Yu et al., 2004). It has been also confirmed that the expression of genes in the cluster is regulated by aflR and aflS. The product of aflR is a Gal 4-type 47-kDa polypeptide which binds to the palindromic sequence 5'-TCGN5CGA-3' in the cluster gene promoters, resulting in transcriptional activation of aflatoxin biosynthesis genes (Alkhayyat & Yu, 2014;Yu et al., 2004). AflS can bind AflR inhibitors and act as a transcriptional enhancer to optimize AflR activity (Alkhayyat & Yu, 2014).

The influence of temperature on aflatoxin biosynthesis by
A. flavus has been investigated in many studies (Bai et al., 2015;Medina et al., 2017;Yu et al., 2011). The optimum temperature for aflatoxin formation is ~30°C, while the optimum temperature for mycelial growth is ~37°C (Yu et al., 2011). To understand the effects of varying temperatures on aflatoxin biosynthesis, several high-throughput technologies, (e.g., transcriptomic and proteomic analyses) have been introduced. Yu et al., (2011) observed a large number of differentially expressed genes between 30°C and 37°C, in mycelia that were harvested 24 hr after inoculation. The average transcription level for the 30 aflatoxin biosynthesis genes increased by ~3,300-fold at 30°C, as compared with 37°C (Yu et al., 2011). Bai et al., (2015) applied transcriptomic and proteomic analyses to identify changes in A. flavus at 37°C for 1.5 days and 28°C for 3 days, showing that post-transcriptional processes play a critical role in regulating the protein level between the two temperatures. Lind, Smith, Saterlee, Calvo, and Rokas (2016) found that 11 temperature-regulated gene clusters, associated with secondary metabolites, were required VeA at 37°C, and LaeA at both 30°C and 37°C. In addition, a large number of gene ontology and KEGG pathways have been identified in maize kernels colonized by A. flavus under different water activities (aw; 0.99 and 0.91) and temperatures (30°C, 37°C) after 10 days (Medina et al., 2017).
With the help of high-throughput technologies, the underlying mechanisms are increasingly being established, although little information is available on the changes in A. flavus mycelial growth and aflatoxin production under low temperature conditions and conflicting results exist on the feasibility of aflatoxin production by A. flavus at 37°C.
In this study, an RNA-Seq approach was used to identify differentially expressed genes (DEGs) and regulatory pathways in A. flavus, under three temperature conditions (20°C, 28°C, and 37°C).
Furthermore, the results of bioinformatic analysis were verified by experiments. The result of this study assists our understanding of the regulatory mechanisms of aflatoxin formation under different temperature conditions and can help develop strategies to control the production of aflatoxins in the food chain.

| Fungal strain and cultivation conditions
An aflatoxin-producing strain, A. flavus NRRL 3357, was provided by Dr. Zhumei He (Sun Yat-sen University, China). This strain can produce high amounts of aflatoxin B1 on YES agar (20 g/L yeast extract, 150 g/L sucrose, 15 g/L agar) under permissive conditions. In order to analyze the mycelial growth and toxin production abilities of A. flavus NRRL 3357 under different temperatures, agar plates were overlaid with sterile 8.5 cellophane sheets and single point inoculated (centrally) with 10 μl of spore suspension (10 6 spores in sterile water) and incubated at 20°C, 28°C, or 37°C. Three biological replicates were used for all subsequent analyses.

| Fungal growth and aflatoxin analyses
Fungal mycelia were collected from the cellophane surface using a scraper, for weight and aflatoxin analyses. All fungal mycelia from each plate were transferred into a 50 ml tube containing 5 ml of methanol at room temperature, then incubated with continual agitation at 150 rpm, for 30 min. The supernatant was collected by centrifugation at 3,000 g and filtered through a syringe filter (RC 0.22 μm, Alltech). The presence of aflatoxin B1 was determined by HPLC with fluorescence detection, using a Waters 600 series HPLC equipped with a 600 pump, a 2,707 autosampler, and a 600 column thermostat set at 30°C. Detection was performed using a 2,475 Multi λ fluorescence detector set at 365 nm (λex) and 465 nm (λem), with a Waters Empower Windows xp operating system (Waters).

| RNA isolation
Total RNA of A. flavus NRRL 3357 was isolated using a RNAiso plus (Takara) according to manufacturer's instructions. The quality and quantity of total RNAs were characterized using an Agilent 2100 and a Nano-Drop 2000c instrument (Thermo Scientific).

| RNA sequencing
Crude RNA was digested using 10 U DNase I (TaKaRa) at 37°C for 30 min. Ribosomal RNAs was removed using TruSeq Stranded

| Real-time quantitative PCR
Real-time quantitative PCR was used to verify the gene expression level calculated from transcriptomic data. DEGs that may regulate fungal growth and aflatoxin production were verified and selected for further investigation. Crude RNA was used to synthesize cDNA using a transScript® first-strand cDNA synthesis superMix kit (Transgen), where the 20 μL reaction system consisted of 10 μl SYBR® Fast qPCR Mix (2x), 0.5 μl of each primer (10 μmol/l) and 1 μl cDNA. The real-time quantitative PCR program was set to the following sequence: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and finally, 60°C for 10 s. The β-tubulin gene was used as an endogenous control, with three biological replicates assessed for each sample. Relative expression levels were calculated using the 2 −ΔΔ CT method.

| Effects of temperature on mycelial growth and aflatoxin production
To assess the effects of temperature on fungal growth and aflatoxin production, three different culture temperatures were assessed. Figure 1a shows that optimal growth of fungal mycelia was observed at 37°C, while the slowest growth occurred at 20°C, as compared with the 28°C control. Cultivation at 28°C resulted in the highest level of aflatoxin B1 production on YES agar, while no aflatoxin B1 was detected by HPLC in all samples grown at 37°C, from 2 days to 7 days ( Figure 1b). Cultivation at 20°C formed significantly less aflatoxin B1 than with that of the 28°C control (p < 0.05) (Figure 1b).
Large differences were observed in both the fungal biomass and aflatoxin B1 content of A. flavus cultured at varying temperatures at 4 days and as changes in gene expression occurs in advance of changes in fungal growth and aflatoxin production, samples at 3 days were selected for RNA-Seq.

| Impact of different temperatures on gene expression
The quality and quantity of total RNAs were examined using Agilent 2100 and Nano-Drop 2000c instruments, confirming that F I G U R E 1 Effects of temperature on A. flavus fungal growth (a) and toxin production (b) the isolated total RNAs were of a good enough quality for cDNA library construction (Table A1 in Appendix 1). Sequencing of all samples yielded a total of 155,177,520 raw paired-end 150-bp reads ( Table A2 in Appendix 1). Assessment of the quality of raw reads by FASTQC showed that an overwhelming majority of reads had quality scores above Q30 (Appendix Figure A1), indicating the quality of the raw reads of all samples could be used for further analyses. 95.37% of reads (147,998,105 paired reads) remained as clean reads, following removal of adaptor, unknown, low quality and rRNA sequences (Table A2 in Appendix 1). The clean reads were used for mapping onto the genome of A. flavus NRRL 3357 for measurement of gene expression levels.

| Identification of DEGs
The genome sequence and related annotation files of A. flavus NRRL 3357 were obtained from the J. Craig Venter Institute (https ://www.jcvi.org/). More than 61% of paired clean reads could be uniquely mapped to the A. flavus NRRL 3357 genome via pipeline HISAT2 (Table A2 in Appendix 1). 81.22% (10,953 out of 13,486) of putative protein-coding genes could be detected throughout all samples and at the cut-off count of ≧ 10 in at least one sample.
Further analysis between samples showed a high correlation of the gene expression levels in the replicates of each treatment ( Figure 2). Due to A. flavus NRRL 3357 producing more aflatoxin at 28°C than at 20°C or 37°C, gene expression following mycelial growth at 28°C was selected as the control. Figure 3 and Table A3 in Appendix 1 show that a total of 2,428 and 1,474 more DEGs were identified in fungal mycelia grown at 20°C and 37°C, respectively, as compared with that of the 28°C control. It is of note that ~79% of the DEGs in 37°C samples belonged to up-regulated genes, while only ~21% of the DEGs were down-regulated. Conversely, ~ 63% of the DEGs in 20°C samples belonged to down-regulated genes (Table A3 in Appendix 1). The Venn diagram shows that 7.1% of DEGs (137 genes) were shared between up-regulated genes, and 4.4% of DEGs (77 genes) were shared between down-regulated genes ( Figure 4).

| GO term and KEGG pathway enrichment analyses of the down-regulated DEGs
The enriched GO terms of the down-regulated DEGs impacted by high temperatures were apparently less than that of lower temperatures. The GO terms of the down-regulated DEGs impacted by lower temperatures were enriched in serine-type carboxypeptidase activity, oxidoreductase activity, nitrate metabolic process, fatty acid catabolic process, catalytic complex, cytoskeletal part, benzenecontaining compound metabolic process, etc, while oxidoreductase activity, aflatoxin biosynthetic process, austinol biosynthetic process, benzene-containing compound metabolic process, protein complex, among others, were the enriched terms under higher temperature conditions ( Figure 5).
KEGG analysis showed that the down-regulated DEGs enriched by lower temperatures were involved in carbon metabolism, nitrogen metabolism, amino acid metabolism, fatty acid degradation, peroxisome, among others ( Figure 6). The enriched pathways of down-regulated the DEGs by higher temperatures were involved in aflatoxin biosynthesis, butanoate metabolism, C5-branched dibasic acid metabolism, biosynthesis of amino acids, etc ( Figure 6). Aflatoxin biosynthesis, butanoate metabolism, oxidation-reduction process, and benzene-containing compound metabolic process were the shared down-regulated pathways between lower and higher temperatures.

| GO term and KEGG pathway enrichment analyses of up-regulated DEGs
The enriched GO terms of the up-regulated DEGs impacted by high temperatures were apparently more than that at lower F I G U R E 2 Correlation of gene expression levels between samples cultured under different temperatures F I G U R E 3 Volcano plots displaying DEGs for two different samples. The y-axis shows the mean expression value of log10 (adjusted p value) and the x-axis displays the log2 fold change value. The significantly up-and down-regulated genes are shown as red dots and blue dots, respectively (p < 0.05, fold change >2). 20vs.-28, mycelia at 20°C compared with control; 37-vs.-28, mycelia at 37°C compared with control temperatures. The GO terms of the up-regulated DEGs impacted by lower temperatures were enriched in iron ion homeostasis, amino acid transport, oxidoreductase activity, monooxygenase activity, carboxylic acid transmembrane transporter activity, steroid metabolic process, cellular protein modification process, oxidation− reduction process, cellular protein modification process, protein complex, etc, while nitrate metabolic process, melanin biosynthetic process, glycosaminoglycan catabolic process, asexual spore wall assembly, O-methyltransferase activity, galactosidase activity, among others were the enriched terms under higher temperature

| Aflatoxin biosynthesis processes
A. flavus aflatoxin biosynthesis genes were first analyzed using the SMURF informatics tool (Khaldi et al., 2010). Thirty genes were annotated in the aflatoxin biosynthetic cluster, with twenty-two aflatoxin biosynthetic genes down-regulated by lower temperatures, while all 30 genes were down-regulated by higher temperatures, as compared to the control conditions (Table 1).

| Oxidoreductase activity
Oxidoreductase activity is driven by laccase or multicopper oxidase.
Although the expression levels of the two genes encoding oxidoreductase activity were significantly higher at 37°C than at 28°C, the total expression levels were ~9% lower at 37°C than at 28°C (Table 2). It is of note, that the total expression level was highest at 20°C, suggesting that oxidation-reduction (redox) reactions are influenced by temperature.

| The DEGs shared by lower and higher temperature
It can be seen that the 77 down-regulated genes shared by lower and higher temperatures were involved in aflatoxin biosynthetic process, and sterigmatocystin biosynthetic process (Table A4 in Appendix 1, Figure 9). The 137 up-regulated genes shared by lower and higher temperatures were involved in cellular macromolecule biosynthetic process, gene expression, nucleic acid metabolic process, and gliotoxin biosynthetic process (Table A5 in Appendix 1, Figure 9). The functions of many DEGs are still unclear.

| Real-time PCR verification and analysis of several DEGs
To verify the reliability of DEGs identified by RNA-Seq, the relative expression levels of several DEGs were further investigated via realtime PCR. Results show that a similar expression pattern was observed between RNA-Seq and real-time PCR data (Appendix Figure   A2), indicating that the relative expression level identified via RNA-Seq was reliable.

| D ISCUSS I ON
The results of the present study demonstrate the complexity of aflatoxin biosynthesis regulation and fungal mycelial growth, under different temperatures. As compared to the control, 1,539 genes were significantly down-regulated by the reduced temperature of 20°C, while only 303 genes were significantly down-regulated by the higher temperature of 37°C, in mycelia at 3 days (Table A3 in Appendix 1). It is also very interesting that a majority of the down-regulated genes at higher temperatures related to secondary metabolic processes ( Figures 5 and 6), while a majority of genes up-regulated by higher F I G U R E 4 Venn diagram depicting the overlap in DEGs shared between up-regulated genes or between down-regulated genes temperatures were related to primary metabolic processes (Figures   7 and 8) related to fungal growth (Wisecaver, Slot, & Rokas, 2014). It also has been established that sterigmatocystin compounds are the precursor substances for aflatoxin synthesis (Georgianna & Payne, 2009 Aflatoxin biosynthesis, butanoate metabolism, oxidation-reduction process, and benzene-containing compound metabolic process were the shared down-regulated pathways, while steroid biosynthesis, oxidoreductase activity, cellular protein modification process, DNA binding, protein complex were the shared up-regulated pathways between lower and higher temperatures. Secondary metabolic process (toxin metabolic process) and oxidoreductase activity were also shared by the absence of two key members (VeA and LaeA) of the Velvet protein complex (Lind et al., 2016). The results of the present study are in accordance with previous investigation, which implied that the enriched pathways in this study should be reliable and can be used for further investigation. The results of the present study also demonstrate that the total expression level of genes related to oxidoreductase activity was lowest at 37°C and highest at 20°C. These results indicate that fast growth mycelia may be less affected by oxidative stress at 37°C, while slow growth mycelia might be affected by stress can cause changes in cytosolic and mitochondrial calcium concentrations in A. nidulans (Greene, Cao, Schanne, & Bartelt, 2002).
Several investigations have also implied that antioxidants can significantly inhibit aflatoxin production, while oxidants enhanced aflatoxin production (Kim et al., 2008;Narasaiah, Sashidhar, & Subramanyam, 2006;Reverberi et al., 2005). According to our results and previously reported studies, oxidative stress resulting from reactive oxygen species might be involved in instigating aflatoxin biosynthesis. The formation of aflatoxin should be regulated via different pathways while being independent from fungal growth.
Previous studies have observed that spore and pigment production is accompanied by the formation of toxins (Chang, 2008;Georgianna & Payne, 2009). In the present study, genes for asexual spore wall assembly were significantly up-regulated under higher temperatures (Figure 7), suggesting that spores are associated or involved in the process of syntheses. Some previous studies have not detected aflatoxins in growth medium at 37°C, while other studies have detected aflatoxins under the same temperatures. According to the enriched pathways, it may be speculated that higher temperatures may block the formation of aflatoxin by delaying the synthesis of its precursor substances during the fast growth stage of fungal mycelia.
According to this theory, we cultivated the fungus again as described in the materials and methods section, incubating samples at 37°C for a longer period of time and at 10 days, aflatoxin concentrations reached as high as 10 μg/g. These results support the proposed theory and explain the contrasting results reported by previous studies.
Several genes have been reported to be involved in the production of aflatoxin. Members of the Velvet protein complex coding genes (VeA, LaeA) and homeobox gene (hbx1) were proved to be required for the formation of aflatoxin (Cary et al., 2019;Lind et al., 2016). All of them were not the down-regulated genes shared by lower and higher temperatures (

CO N FLI C T O F I NTE R E S T S
The authors declare no conflict of interest.

AUTH O R CO NTR I B UTI O N S
Fang Tao conceived and designed experiments. Guomin Han carried out the bioinformatics studies, and Kai Zhao conducted experiments. Guomin Han and Fang Tao wrote the manuscript. Xiaodan Yan, Fangzhi Xiang, and Xuede Li helped draft the manuscript.

E TH I C S S TATEM ENT
None required.

DATA AVA I L A B I L I T Y S TAT E M E N T
The raw paired-end sequence data are available in SRA under accession number SRP159671, https ://www.ncbi.nlm.nih.gov/sra/SRP15 9671.