Aspergillus flavus SUMO Contributes to Fungal Virulence and Toxin

Article pubs.acs.org/JAFC

Aspergillus flavus SUMO Contributes to Fungal Virulence and Toxin Attributes Xinyi Nie, Song Yu, Mengguang Qiu, Xiuna Wang, Yu Wang, Youhuang Bai, Feng Zhang, and Shihua Wang* Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China S Supporting Information *

ABSTRACT: Small ubiquitin-like modifiers (SUMOs) can be reversibly attached to target proteins in a process known as SUMOylation, and this process influences several important eukaryotic cell events. However, little is known regarding SUMO or SUMOylation in Aspergillus flavus. Here, we identified a novel member of the SUMO family in A. flavus, AfSumO, and validated the existence of SUMOylation in this pathogenic filamentous fungus. We investigated the roles of AfsumO in A. flavus by determining the effects of AfsumO mutations on the growth phenotype, stress response, conidia and sclerotia production, aflatoxin biosynthesis, and pathogenicity to seeds, and we found that SUMOylation plays a role in fungal virulence and toxin attributes. Taken together, these results not only reveal potential mechanisms of fungal virulence and toxin attributes in A. flavus but also provide a novel approach for promising new control strategies of this fungal pathogen. KEYWORDS: AfsumO, SUMOylation, Aspergillus flavus, morphogenesis, aflatoxin, pathogenicity

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INTRODUCTION Aspergillus flavus, a saprotrophic and pathogenic filamentous fungus found in soils worldwide, is notorious for its colonization of several important crops, such as maize, peanut, cottonseed, and rice, in the field or during harvest, storage, and transport.1−3 A. flavus infections can cause the crops to rot, which reduces the grade and price of the grains and also exposes a wide variety of food or feed commodities to the polyketide-derived carcinogenic and mutagenic mycotoxins, commonly known as aflatoxins.2,4 Estimates from the Food and Agriculture Organization indicate that nearly 25% of the world’s grains are contaminated by mycotoxins, of which the most notorious are aflatoxins.1 This contamination leads to enormous agricultural economic losses1,5 and poses a serious threat to both food safety and the health of humans and domestic animals.1,2,6 Therefore, measures to restrict the virulence of A. flavus, including its colonization and aflatoxin production, are urgently needed, not only to prevent agricultural economic losses and ensure food safety but also to protect human and animal health. Clarification of the molecular bases underlying the fungal virulence and toxin attributes is critical to the development of rational control strategies for A. flavus. Despite numerous studies regarding the genetic and environmental factors influencing the growth, development, and secondary metabolism of A. flavus (as reviewed by Amare and Keller7), many aspects related to its fungal pathogenesis are still poorly understood. In the past decade, post-translation modifications, such as phosphorylation,8,9 ubiquitination,10,11 and SUMOylation,12−14 have been well documented as providing an important link between genotype, environmental influence, and phenotype in most eukaryotic organisms. However, there has been little progress in addressing post-translation modifications in A. flavus. As the modifiers in SUMOylation, small ubiquitin-like modifiers (SUMOs) can be reversibly attached to target proteins © 2016 American Chemical Society

and subsequently modulate their fate as well as their subcellular location and function.15,16 Consequently, SUMO can be involved in many cellular processes, such as cell cycle progression, genome stability, DNA repair, and stress response.17−19 In contrast to the numerous studies regarding SUMO in vertebrates, plants, and yeast, there have been only three studies related to SUMO in Aspergillus spp. over the past decade.20−22 The deletion of the single SUMO-coding gene in Aspergillus nidulans not only resulted in the exhibition of impaired growth, the reduction of conidiation, and selfsterility20,22 but also dramatically altered the production of certain secondary metabolites.21 Accordingly, it seems likely that SUMO also plays an important role in fungal biology and mycotoxin biosynthesis in A. flavus. Nevertheless, there are no reports of a SUMO homologue or on the role of SUMOylation in A. flavus. Whether or not SUMO and its modification are relevant for the fungal biology and aflatoxin biosynthesis of this important filamentous fungus is still unknown. Here, we identified a member of the SUMO family that performs SUMOylation in A. flavus. We then investigated the roles of the SUMO gene and of SUMOylation in A. flavus by determining the effects of a mutation in the SUMO-encoding gene on the growth phenotype, conidia, and sclerotia production, aflatoxin biosynthesis, and pathogenicity on seeds of A. flavus. Our main objective was to gain insight into the cross-talk between SUMOylation and the virulence and toxin attributes of A. flavus. Received: Revised: Accepted: Published: 6772

May 15, 2016 August 9, 2016 August 17, 2016 August 17, 2016 DOI: 10.1021/acs.jafc.6b02199 J. Agric. Food Chem. 2016, 64, 6772−6782

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Journal of Agricultural and Food Chemistry Table 1. Aspergillus flavus Strains Used in This Study

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strain

genotype

source

CA14 Wild type gpdA(p)-mCherry-AfsumO gpdA(p)-mCherry ΔAfsumO OE::AfsumO

pyrG-, niaD-, Δku70 niaD-, Δku70 pyrG-, gpdA(p)::mCherry::AfsumO CDS::AfsumO(t)::pyrG, niaD-, Δku70 pyrG-, gpdA(p)::mCherry::AfsumO(t)::ptrA, niaD-, Δku70 pyrG-, ΔAfsumO::pyrG, niaD-, Δku70 pyrG-, gpdA(p)::AfsumO:: pyrG, niaD-, Δku70

ref24 ref24 this study this study this study this study

secondary antibody was added, and ECL reagent was used for detection by G:BOX Chemi XT4 (Syngene, UK). Measurement of Conidia and Sclerotia Production. About 103 spores was seeded at the center of each Petri dish (60 × 15 mm) of PDA or Wickerham medium agar. For quantitative comparison of the production of conidia and sclerotia, cultures in triplicate were grown in the dark or under white light at 29 or 37 °C for relevant days. Four 7 mm diameter agar plugs from the center along the radius of each plate were cored with a plastic transfer pipet. The plugs were placed in a 2 mL microcentrifuge tube with 1 mL of 0.05% Tween-20 solution. Conidia were collected and counted on a hemocytometer. Sclerotia were counted manually. Aflatoxin Extraction from YESSB and High-Performance Liquid Chromatography (HPLC). Spores were inoculated onto the Petri dish plate (100 × 15 mm) containing 20 mL of YESSB and led to the final concentration of 105 spores/mL. Cultures were incubated at 29 °C in the dark for 5 days. Metabolites from the 0.2 mL stationary cultures were extracted with 0.2 mL of chloroform. Aliquots of the organic layer were transferred to clean microcentrifuge tubes and airdried. The aflatoxin extract was filtered (0.22 μm) and analyzed by Breeze HPLC (Waters, USA) on a MYCOTOX reversed-phase C18 column (4.6 × 250 mm, Pickering Laboratories, USA) at 42 °C. The column was equilibrated in the running solvent (methanol/acetonitrile/ water = 22:22:56), and 10 μL was injected and run isocratically for 14 min with 100% running solvent at a flow rate of 1.0 mL/min. Aflatoxin was detected by a fluorescent detector with an excitation wavelength of 365 nm and an emission wavelength of 430 nm. The identification was confirmed by co-injection with a commercially available standard. Real-Time Fluorescence Quantitative Reverse Transcription PCR (qPCR). Mycelium was harvested after 72 h of incubation in YESSB medium at 29 °C in the dark, immediately fully ground in liquid nitrogen, and stored at −80 °C for RNA extraction. RNA was isolated from 100 mg of ground mycelium using the Eastep Total RNA Extraction Kit (Promega, USA) and treated with RNase-free DNase I (ThermoFisher Scientific, USA). Subsequently, cDNA was synthesized from RNA using the RevertAid First Strand cDNA Synthesis Kit (ThermoFisher Scientific, USA). qPCR was performed on a PikoReal Real-Time PCR machine (ThermoFisher Scientific, USA) using the SYBR Green qPCR mix (TaKaRa Biotechnology, Japan) (primers listed in Table S2). The 18S rDNA gene was used as the endogenous assay control. REST 2009 software was used to calculate the relative expression of target genes using the PairWise Fixed Reallocation Randomization Test.25 Seed Infections, Aflatoxin Extraction, and Thin Layer Chromatography (TLC). The procedures of seed infection and aflatoxin extraction from peanuts and maize followed previously described methods.26 The extracts were air-dried and then resuspended in 500 μL of chloroform, and 10 μL of each extract was separated on a silica gel TLC plate using the chloroform/acetone (80:20 v/v) solvent system. Statistical Analysis. All data were presented as the means ± standard deviation (SD). The presence of statistical differences among groups was determined by one-way ANOVA, and the method of least significant difference (Dunnett’s multiple-comparisons test) was used to compare the effects between each group and the control. All of the statistical analyses were carried out using the statistical software SPSS 13.0 for Windows. Statistical significance was recognized when P < 0.05.

MATERIALS AND METHODS

Chemicals. Aflatoxin B1 standard, Calcofluor White (CFW), Congo-Red (CR), diamide, 4′,6-diamidino-2-phenylindole (DAPI), hydroxyurea (HU), and menadione sodium bisulfite (MSB) were obtained from Sigma-Aldrich (USA), with purity >95%. Methylmethanesulfonate (MMS) was bought from Amresco (USA). Anti-mCherry and anti-β-actin antibodies were obtained from Abcam (UK). Horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG antibody and the immobilon western chemiluminescent HRP substrate were bought from Merck Millipore (USA). All other chemicals were of analytical grade and obtained from commercial sources. Strains and Culturing Conditions. For the preparation of plasmid DNA, the Escherichia coli strain JM109 was used. A. flavus strains used in this study are listed in Table 1. Potato dextrose broth (PDB, BD Difco, USA) was used for growth of mycelium, and potato dextrose agar (PDA, BD Difco, USA) was used for growth and conidiation of Aspergillus strains, supplemented with the appropriate amounts of uridine (5 mM), uracil (5 mM), or pyrithiamine (100 ng/mL) when necessary. Modified Wickerham medium23 was used for sclerotia production. YESS broth (YESSB, 2% yeast extract, 15% sucrose, 1% soytone, pH 5.5) was used for aflatoxin production. Mutant Strains Construction. The gpdA(p)-mCherry-AfsumO strain was constructed as previously20 besides the replacement of AfsumO promoter with A. nidulans gpdA promoter. Primers used are listed in Table S1. The red fluorescent protein tag encoding fragment, mCherry, was amplified from plasmid pmCherry-C1, which was a kind gift from Dr. Bowen Li (Fujian Institute of Research on Structure of Matter, Chinese Acadamy of Sciences). The control strain gpdA(p)mCherry was constructed by transformation of the plasmid pPTRIgpdA(p)-mCherry. Three fragments carrying A. nidulans gpdA promoter, mCherry coding sequence, and AfsumO terminator were separately amplified and fused by PCR (primers listed in Table S1) and inserted into the plasmid pPTR I (TaKaRa Biotechnology, Japan). For ΔAfsumO construction, a 3.7 kb AfsumO deletion cassette carrying AfsumO upstream fragment and A. fumigatus pyrG and AfsumO downstream fragment was created by fusion PCR and transformed into A. flavus CA14.24 To create the AfsumO overexpression cassette, four PCR fragments carrying AfsumO upstream fragment, A. fumigatus pyrG, A. nidulans gpdA promoter, and AfsumO gene fragment were fused as described above and transformed into A. flavus CA14. Southern blotting and semiquantitative reverse transcription PCR were conducted to confirm the construction of two mutant strains. Confocal Laser Scanning Tomography. Mycelium from the overnight PDB culture was fixed with 70% ethanol for about 1 h at room temperature, washed with phosphate-buffered saline, and incubated in 1 μg/mL DAPI for 15 min. A coverslip was mounted and imaged using Leica SP8 confocal laser scanning microscope. Dual-channel imaging was used to sequentially image cells labeled with DAPI (excitation, 405 nm; emission bandwidth, 420−460 nm) and mCherry (excitation, 552 nm; emission bandwidth, 600−630 nm). Immunoblotting Analysis. To extract the total cellular protein, the mycelium was fully ground to powder in liquid nitrogen and dissolved in ice-cold RIPA buffer (Beyotime, China). The total lysate was subjected to a NuPAGE Novex Tris−acetate gel electrophoresis system (Life Technologies, USA). After transfer to a PVDF membrane, the membrane was blocked with 5% skim milk powder in Tris-buffered saline and was incubated with antibody against target protein, overnight at 4 °C. After extensive washing, horseradish peroxidase-conjugated 6773

DOI: 10.1021/acs.jafc.6b02199 J. Agric. Food Chem. 2016, 64, 6772−6782

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Journal of Agricultural and Food Chemistry

Figure 1. Sequence analysis of the SumO-predicted peptide in A. flavus. (A) Alignment of the A. flavus SumO-predicted peptide (Af_SumO) sequence with its homologues from A. nidulans (An_SumO), S. cerevisiae (Sc_Smt3), Candida albicans (Ca_Smt3), S. pombe (Sp_Pmt3), Drosophila melanogaster (Dm_Smt3), Xenopus laevis (Xl_SUMO1), and Homo sapiens (Hs_SUMO1). Identical residues present in at least half of the sequences are indicated by the colorful boxes. Sequences were aligned using DNAMAN software. The conserved diglycine motif required for SumO conjugation to target proteins is boxed. (B) Phylogenetic tree of A. flavus SumO predicted peptide and its homologues based on the homology of their primary sequences. The tree was constructed using DNAMAN software with 1000 bootstrap replicates.

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RESULTS Identification of the SUMO Homologue in A. flavus. A single encoding sequence (AFL2G_07682.2), designated AfsumO, was obtained from Aspergillus Comparative Database at Broad Institute (https://www.broadinstitute.org/scientificcommunity/sci-ence/projects/fungal-genome-initiative/ aspergillus-genome-projects). Two introns (from +79 to +171 and from +451 to +518) were revealed by comparison of the predicted AfsumO genomic DNA and cDNA sequences. The AfsumO open reading frame encodes a predicted protein of 92 amino acids, referred to as AfSumO, with a molecular mass of approximately 10.2 kDa. Comparisons of amino acid sequences revealed that AfSumO shows the highest similarity (94%) to the orthologue in another Aspergillus member, A. nidulans, and is also highly similar to previously reported fungal SUMO orthologues, such as Saccharomyces cerevisiae Smt3, Candida albicans Smt3, and Schizosaccharomycs pombe Pmt3. The diglycine residues motif required for target attachment was conserved among AfSumO and its homologues, but AfSumO lacked the C-terminal stretch of amino acids that mask the diglycine motif in previously identified SUMOs (Figure 1A). A phylogenetic analysis was performed with the amino acid sequences of the AfSumO peptide and the previously reported SUMO orthologues, and the results revealed the relationship among them (Figure 1B). Validation of SUMOylation in A. flavus. We speculated that AfSumO, like the other SUMO orthologs, exerts its function through its covalent, post-translational attachment to target

proteins. To confirm that AfSumO can be conjugated to cellular proteins in vivo, immunoblotting and subcellular localization assays for AfSumO and potential AfSumO-conjugated proteins were performed. As the commercially available antibody specific for S. cerevisiae Smt3 did not cross-react with the homologue in A. flavus (data not shown), an AfsumO mutant strain with a fusion tag for detection needed to be constructed. mCherry is a monomeric fluorescent protein that demonstrates both low cytotoxicity and minimal influence on fusion proteins constructed with it,27 which make it a suitable fusion partner for many proteins of interest.28−30 Additionally, as the C-terminus of SUMO is required for its attachment to substrate proteins, an Nterminal-fused tag was more suited for use in an analysis of SUMO−protein conjunction.19,31,32 Accordingly, a mutant strain carrying an mCherry-AfsumO fusion gene under the control of the stronger A. nidulans gpdA promoter and its reference strain with a randomly integrated gpdA(p)-mCherry cassette were generated for both immunoblotting and fluorescence imaging analysis. The strategy for their construction is demonstrated in Figure 2A. As shown in Figure 2B, an immunoblotting analysis using antimCherry antibodies detected free mCherry-AfSumO and a variety of mCherry-AfSumO-conjugated proteins in crude extracts prepared from the mycelium of the gpdA(p)-mCherryAfsumO strain, which closely resembled the SUMOylation patterns detected by others.20,22,33 The subcellular distribution of AfSumO and sumoylated proteins in the mycelium of the gpdA(p)-mCherry-AfsumO strain was investigated by using 6774


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Figure 2. AfSumO−protein conjugation in A. flavus. (A) Diagrammatic representation of the gene replacement strategy for construction of the gpdA(p)mCherry-AfsumO strain. (B) Immunoblotting of 50 μg of crude protein extracts from the wild type, gpdA(p)-mCherry, and gpdA(p)-mCherry-AfsumO strains. (C) SUMOylation pattern in gpdA(p)-mCherry-AfsumO strain under different temperatures. (D) Confocal laser scanning images of the gpdA(p)mCherry-AfsumO stain and the control strain (gpdA(p)-mCherry). Scale bar = 10 μm. All strains were incubated in PDB medium at 29 °C (panels B−D) or 37 °C (panel C) for 20 h. Gel electrophoresis systems in panels B and C were carried out using the 7 and 3−8% NuPAGE TA Gel Systems, respectively.

we generated AfsumO deletion and overexpression mutant strains for loss-of-function and gain-of-function assays, respectively. The strategies for the construction of these mutant strains are illustrated in Figure 3A,B. Southern blotting and semiquantitative reverse transcription PCR were conducted to confirm the construction of these two mutant strains, which are referred to in the present study as ΔAfsumO and OE::AfsumO for the AfsumO deletion and overexpression mutant strains, respectively (Figure 3C,D). To examine if AfSumO mediates the growth of A. flavus, colony growth phenotypes of the AfsumO mutant strains were determined on complete medium (Figure 4). Neither mutation significantly affected the size of the colony on PDA medium at 29 or 37 °C in the absence of cell stress stimuli. The stress growth

confocal scanning laser microscopy (Figure 2D). It was observed that mCherry-tagged AfSumO and its substrates were not only distributed in the cytoplasm but also highly concentrated in the nucleus. In contrast, mCherry signals in the reference strain were detected mainly in cytoplasmic locations. Furthermore, an immunoblotting assay was performed using protein extracts prepared from the mycelium of the gpdA(p)mCherry-AfsumO strain grown at different temperatures, and an increase in the amount of modified mCherry-AfSumO substrates and a decrease in the amount of free mCherry-AfSumO were observed at 37 versus 29 °C (Figure 2C), which implies that SUMOylation in A. flavus is temperature-dependent. Effects of AfsumO Mutations on Growth and Stress Response. To investigate the impacts of AfSumO on A. flavus, 6775


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Figure 3. Construction of the AfsumO deletion (ΔAfsumO) and overexpression (OE::AfsumO) strains. (A, B) Scheme of deletion and overexpression strategy for AfsumO, respectively. P indicates the cutting site of PvuII. (C) Southern blot analysis of the wild type, ΔAfsumO, and OE::AfsumO strains. DNA size for each hybridization band is shown on the right. Genomic DNA from each strain was digested with PvuII and hybridized with a 1.1 kb probe of the upstream region fragment of AfsumO, which was amplified by primers RSP2 and sumOprobR as indicated in panels A and B. (D) Semiquantitative PCR to detect the AfsumO gene transcript levels in the wild type, ΔAfsumO, and OE::AfsumO strains. 18s rDNA was used as the endogenous assay control.

condition, however, had a noticeable effect on the radial growth of the AfsumO mutant strains. When grown at 29 °C, the ΔAfsumO strain was more sensitive than the wild type to MMS, but at 37 °C, this strain also exhibited more sensitivity than the wild type to HU, t-BOOH, and H2O2 as well as to MMS. In contrast, the OE::AfsumO strain had similar or slightly enhanced levels of stress tolerance compared with the wild type, confirming that the change of stress sensitivity was specific to the loss of AfsumO. Effects of AfsumO Mutations on Conidia and Sclerotia Production. The conidiation and sclerotia productions of the AfsumO mutant strains were examined under different conditions. The deletion of the AfsumO gene led to a suppressive effect on conidiation, regardless of temperature and illumination conditions (Figure 5A,C,E). When grown at 29 or 37 °C, the amount of conidia produced by the ΔAfsumO strain was about 50 or 75%, respectively, of the wild type level (Figure 5A,C). In contrast, the OE::AfsumO strain had comparable or denser conidiation than did the wild type (Figure 5A,C). Furthermore, light microscopy observations of the wild type and the AfsumO

mutant strain conidiophores also revealed a visual difference in the conidiation among them (Figure 5E). Notably, the two AfsumO mutations had temperaturedependent biphasic regulation effects on sclerotia production in A. flavus (Figure 5B,D). The ΔAfsumO strain had active sclerotial formation when grown on sclerotia-conducive Wickerham medium at 29 °C, producing >1-fold more sclerotia than the wild type did in the dark. However, when the growth conditions of the cultures were switched to 37 °C, we found that the deletion of AfsumO had an adverse effect on the sclerotia production at this temperature, with the ΔAfsumO strain producing amounts that were only about 50% of those produced by the wild type strain. Differences in the sclerotia production caused by the loss of AfsumO were inverse in the OE::AfsumO strain under the corresponding conditions. Effects of AfsumO Mutations on Aflatoxin Biosynthesis and the Expression of Relevant Genes. In our preliminary experiments, when the ΔAfsumO strain was grown on either PDA or Wickerham media, we observed changes in the color of the media (data not shown). This finding supports the idea that 6776


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the entire surface of the host seeds by the wild type and OE::AfsumO strains, the ΔAfsumO strain showed a delayed and poor conidiation and, moreover, grew aerially with fluffy hyphae (Figure 7A,D). The visual results were also reflected by the conidial counts; the amount of conidiation on seeds was much lower in the ΔAfsumO strain and was significantly higher in the OE::AfsumO strain as compared with that in the wild type strain (Figure 7C,F). The aflatoxin contamination in colonized seeds was subsequently assessed by TLC analyses. As shown in Figure 7B,E, the ΔAfsumO strain produced less AFB1 on both peanut and maize seeds than did the wild type, in contrast to the higher levels of AFB1 production by the OE::AfsumO strain grown on seeds. These results are consistent with the observed effects of the AfsumO mutations on aflatoxin biosynthesis in YESSB medium.

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DISCUSSION As a member of the ubiquitin-like protein family, the spatial structure of SUMO resembles that of ubiquitin.34,35 SUMO plays regulatory roles in several important cell events, such as protein localization and protein−protein/DNA interaction, through its reversible, post-translational conjugation to target proteins, commonly known as SUMOylation. A SUMO-encoding gene and the process of SUMOylation were initially discovered in S. cerevisiae,36 and these have since been identified extensively in higher eukaryotes.37−39 However, studies on SUMOylation and SUMO homologues in Aspergillus spp. are still rare, and no previous publications have yet reported the existence of these in A. flavus. Here, we identified a single homologue of the SUMO gene in the A. flavus genome, which we designated AfsumO. Comparisons of amino acid sequences revealed that the diglycine residues corresponding to the C-terminal of mature SUMO were completely conserved among AfSumO and its orthologues (Figure 1). However, AfSumO lacks a C-terminal stretch of amino acids, and, therefore, has an exposed diglycine motif in its C-terminus, which differentiates it from the previously identified SUMO orthologues. To date, all of the reported SUMO proteins carry a conserved diglycine residue motif, which is essential for the conjunction between a mature SUMO peptide and the enzymes that catalyze SUMOylation, as well as a C-terminal stretch of 2−11 amino acids that masks the diglycine motif.40 It has been widely held that the removal of this C-terminal stretch by a cysteine-specific SUMO protease (e.g., ULP in yeast and SENP in mammals) to expose the diglycine motif is a prerequisite for SUMOylation.41 By searching in the public genome database, in addition to AfSumO, we also found a few similar hypothetical SUMO proteins in other Aspergillus spp. (Table S3) that might be expressed without any amino acids after the diglycine motif. However, whether or not these unusual homologues can still exert a function like other previously identified SUMO peptides remains to be determined. The finding that AfSumO can be conjugated to cellular proteins in vivo like previously reported SUMO orthologues (Figure 2) not only establishes the existence of SUMOylation in A. flavus, but also, for the first time, demonstrates a novel functional SUMO orthologue that is directly expressed as a mature peptide rather than as an inactive precursor. Further studies should aim to determine the difference between this novel SUMO and the other known SUMO orthologues. During recent years, SUMO orthologues and SUMOylation have been documented to play important roles in increasingly

Figure 4. Growth of the wild type, OE::AfsumO, and ΔAfsumO strains on PDA medium only or with various stress stimuli, including 0.01% methylmercuric sulfate (MMS), 10 mM hydroxyurea (HU), 0.5 mM tert-butylhydroperoxide (t-BOOH), 6 mM hydrogen peroxide (H2O2), 0.12 mM menadione sodium bisulfite (MSB), 1% ethanol (EtOH), 2 mM diamide, 1 M NaCl, 200 μg/mL Congo-Red (CR), 80 μg/mL Calcofluor White (CFW), and 100 μg/mL sodium dodecyl sulfate (SDS). Three microliters of 10-fold serially diluted spores (107−104/ mL) were spotted on the PDA plates and then incubated for 36 h at 29 °C (A) or for 24 h at 37 °C (B).

AfsumO modulates the A. flavus secondary metabolism. Thus, we examined the strains to investigate possible effects of AfsumO expression on aflatoxin production. The metabolite extracts from strains that were cultured in YESSB for 5 days at 29 °C in the dark were assessed by HPLC analysis. The results demonstrated that both levels of the AFB1 and AFB2, the two major aflatoxins produced by A. flavus, severely decreased in the ΔAfsumO strain, but increased nearly 100% in the OE::AfsumO strain, as compared with the wild type (Figure 6A,B). Subsequently, we measured the levels in these strains of the transcripts for genes relevant to aflatoxin biosynthesis (Figure 6C). The results of a qPCR analysis indicate that the disruption of AfsumO impairs the expression of most candidates, including the gene encoding the upstream global transcription factor of the aflatoxin biosynthesis pathway, af lR, the accessory regulatory gene, af lS, and several genes for key downstream enzymes, such as af lA, af lC, af lD, af lO, and af lP.7 In contrast, compared with the wild type, much more mRNA produced by these biosynthesis-relevant genes was detected in the OE::AfsumO strain. These qPCR results correlate with aflatoxin production. Pathogenicity of the AfsumO Mutant Strains on Seeds. To verify the pleiotropic effects of the AfsumO mutations that we observed on synthetic media, we examined and contrasted the virulence and toxin attributes of the AfsumO mutant strains on both peanut and maize seeds. Visually, the strain lacking AfsumO was crippled in its ability to colonize and sporulate on host seeds. After 5 days of inoculation, in contrast to the full colonization on 6777


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Figure 5. Morphological phenotype analysis of AfsumO deletion and overexpression strains on conidia and sclerotia production. (A) Colony morphology of the wild type, ΔAfsumO, and OE::AfsumO strains on PDA medium under indicated growth conditions. (B) Colony morphology (after washing off the conidia) of the different strains on Wickerham medium under indicated growth conditions. (C, D) Amounts of conidia and sclerotia produced by the different strains, respectively. (E) Conidiophores of the wild type and AfsumO mutant strains by light microscope. Values are a mean of four replicates. ∗ and ∗∗ indicate significant difference between the wild type and mutant strains at P < 0.05 and P < 0.01, respectively, as assessed by oneway ANOVA and Dunnett’s multiple-comparisons test.

more fungi, such as S. cerevisiae,36,42 S. pombe,33,43 C. albicans,44 and A. nidulans.20−22 Here, we investigated the effects of AfsumO mutations on the fungal biology and aflatoxin biosynthesis of A. flavus. First, we found that SUMOylation in A. flavus might be induced at 37 °C. It has become clear that SUMOylation plays important roles in the cellular stress response, including heat

shock,19,38,39 and many cellular stresses lead to increased formations of SUMO attachments. However, in the present study, we did not find that the growth phenotype of the AfsumO mutant strains was sensitive to temperature change (Figure 4). Thus, it seems likely that the elevated level of SUMOylation at 37 °C could be due to temperature-sensitive modulations in the 6778


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Figure 6. Aflatoxin production and aflatoxin biosynthesis-relevant gene expression in AfsumO deletion and overexpression strains. (A) HPLC profiles of metabolite extracts for aflatoxin production of wild type, ΔAfsumO, and OE::AfsumO strains. (B) Relative levels of aflatoxins B1 and B2 calculated from the HPLC analysis in part A; three independent isolates of each strain were analyzed. ∗∗ indicates significant difference between the wild type and mutant strains (P < 0.01), as assessed by one-way ANOVA and Dunnett’s multiple-comparisons test. (C) Transcript levels for nine aflatoxin biosynthesis genes in the wild type, ΔAfsumO, and OE::AfsumO strains. Each bar indicates the mean ± standard deviation (SD) of four replicate assay experiments. ∗ and ∗∗ indicate significant difference between the wild type and mutant strain at P < 0.05 and P < 0.01, respectively, as assessed according to randomization test (REST software). All strains were cultured in YESSB medium for 72 h at 29 °C.

distribution of AfSumO (Figure 2D). This finding is similar to those reported for the deletion of SUMO in S. pombe33 and A. nidulans,20 and it suggests that there is an overlap between SUMOylation and the signaling pathway to protect against DNA damage challenges. The AfsumO deletion mutant also exhibited observable effects on both sporulation and sclerotia production. Compared with its temperature-dependent biphasic regulation effects on sclerotia

enzymatic activities of the SUMOylation cascade. The temperature-sensitive stress response and sclerotial production observed in this work might also be accompanying effects of the elevated level of SUMOylation. The fact that the ΔAfsumO strain was strikingly sensitive to the DNA damage agent MMS indicates that Af SumO might be essential for DNA damage response and subsequent repair, an idea that is also indirectly supported by the observed subcellular 6779


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Figure 7. Pathogenicity of AfsumO deletion and overexpression strains: morphology of fungal colonies on living peanut cotyledons (A) or maize (D) after 5 days of inoculation; thin layer chromotrography measurements of aflatoxin B1 extracted from peanut cotyledons (B) or maize (E) after 5 days of inoculation (“AFB1 → ” indicates the aflatoxin B1 standard); conidia production on peanut cotyledons (C) or maize (F) after 5 days of inoculation. ∗ and ∗∗ indicate significant difference between the wild type and mutant strain at P < 0.05 and P < 0.01, respectively, as assessed by one-way ANOVA and Dunnett’s multiple-comparisons test.

significantly lower in the ΔAfsumO strain than that in the wild type strain. Simultaneously, the transcription of genes relevant to aflatoxin production, including several structural genes and their upstream regulator genes, af lR and af lS,7 were prominently

production, the deletion of AfsumO resulted in a more specific adverse effect on conidiation (Figure 5), which is consistent with the effect of SUMO deletion in A. nidulans.20 Additionally, the biosynthesis of aflatoxins, including AFB1 and AFB2, was 6780


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Journal of Agricultural and Food Chemistry suppressed in the ΔAfsumO strain (Figure 6). Therefore, it could be suggested that AfSumO or SUMOylation plays an important role in activating the aflatoxin biosynthesis-relevant gene cluster. A. flavus has the potential to colonize on oilseed crops by sporulation on injured seeds and, subsequently, to contaminate the hosts with aflatoxin. Although the physiological significance of these SUMOylation events remains unknown, on the basis of the reduction of conidiation and aflatoxin biosynthesis as a result of the inactivation of AfsumO, AfSumO or SUMOylation may be relevant to fungal virulence and toxin attributes of A. flavus (Figure 8). This idea is further supported by the pathogenic phenotypes of the AfsumO mutant strains on both peanut and maize seeds (Figure 7).

In conclusion, we identified a novel and functional SUMO orthologue in A. flavus, and we determined the effects of SUMOylation by AfSumO on A. flavus fungal biology and aflatoxin biosynthesis. Our preliminary results suggest potential mechanisms underlying the fungal virulence and toxin attributes of A. flavus, and they provide a novel prospect for developing new fungal control strategies. However, further investigation is required to discover the components of the SUMOylation machinery in A. flavus and to determine the molecular mechanism of SUMOylation cross-talk with other important signal pathways.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b02199. Tables S1−S3 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(S.W.) Phone/fax: +86-591-87984471. E-mail: wshyyl@sina. com. Funding

This work was funded by the National Basic Research Program of China (973 Program, No. 2013CB127802), the Natural Science Foundation of Fujian Province (No. 2016J05066), the Education Department of Fujian Province (No. JA15170), and the Fujian Agriculture and Forestry University (No. KF2015045). Notes

The authors declare no competing financial interest.

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REFERENCES

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Figure 8. Schematic diagram illustrating putative mechanisms by which AfSumO or SUMOylation cross-talks with A. flavus virulence and toxin attributes.

Coping with the severe agricultural impacts of A. flavus can be achieved by either preventing fungal infection or restricting aflatoxin biosynthesis. Although a large number of genetic and environmental factors relevant to fungal virulence and aflatoxin production have been reported, unfortunately, no effective and economical control strategies based on these factors have been created yet.2,7,45 Over the past few years, some studies have begun to pay attention to the potential of SUMOylation as a drug or treatment target in various human diseases ranging from neurodegenerative disorders to metabolic syndromes.46−48 Targeting the SUMOylation processing enzymatic cascade has been proposed as a novel approach for “next-generation” clinical therapies.48−50 On the basis of the roles of SUMOylation in the virulence and toxin attributes of A. flavus demonstrated by our work, the exploitation of active small molecule inhibitors aimed at SUMOylation processing enzymes or substrates may lead to new promising control strategies for A. flavus. 6781


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Aspergillus flavus SUMO Contributes to Fungal Virulence and Toxin

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