Toxins, Vol. 14, Pages 883: Putative C2H2 Transcription Factor AflZKS3 Regulates Aflatoxin and Pathogenicity in Aspergillus flavus

1. IntroductionA characteristic of fungi is the ability to produce a wide variety of secondary metabolites, including beneficial compounds such as lovastatin, as well as toxic molecules such as mycotoxins [1]. Aspergillus flavus, a conditional fungal pathogen of important crops in pre- and post-harvest periods, produces carcinogenic aflatoxins (AFs) that cause severe yield reduction and represent a serious threat to animal and human health [2]. A study by the Food and Agriculture Organization proved that about a quarter of the world’s total food production is contaminated by mycotoxins each year, and the main source of pollution is A. flavus and its secondary metabolites [3]. Therefore, exploring the complex mechanism and regulatory network of AF biosynthesis will help to develop effective measures to control the growth of A. flavus and AF contamination, protecting human and animal health and reducing huge economic losses to agricultural production.The biosynthesis of AFs is regulated by global and pathway-specific transcription factors. Pathway-specific transcription factors, including aflR and aflS within the AF gene cluster, have been studied extensively [4]. AflR is a DNA-binding zinc cluster protein that binds to a palindromic sequence in the promoter region to activate gene expression [5,6]. AflR is necessary for AF synthesis, and the deletion of aflR leads to the downregulation of genes and the complete loss of AF synthesis [7]. AflS regulates genes in the AF synthesis gene cluster by assisting the localization of aflR [8]. Additionally, the biosynthesis of AFs is also regulated by global transcription factors such as zinc finger, bZIP, PHD, homeobox, and APSES transcription factors [9,10,11,12]. Among them, the zinc finger family is the largest and includes the Cys2His2 (C2H2), Cys4 (C4), and Zn(Ⅱ)2C6 subfamilies [13]. Researchers have identified some zinc finger transcription factors with global regulatory functions. The transcription factors nsdC and nsdD, essential for the development of A. nidulans, are also involved in the growth and development of A. flavus, as well as secondary metabolism. AF production is completely lost in nsdC-deleted strains, and aflD, aflM, and aflP genes are not expressed [14]. MtfA encodes a C2H2 zinc finger transcription factor that influences the production of sterigmatocystin, and the overexpression of mtfA can dramatically decrease secondary metabolites such as AFB1 [1]. RsrA, a highly conserved C2H2 transcription factor in A. nidulans, regulates the synthesis of sterigmatocystin, a precursor of AF [15]. These results suggest that C2H2 transcription factors play regulatory roles in mycelia growth development and secondary metabolism. Genome annotation (http://ftfd.snu.ac.kr/index.php?a=view, accessed on 10 October 2022) revealed a putative C2H2 zinc finger transcription factor encoded by AflZKS3 in the genome of A. flavus, which shares 83% homology with the IFM54703_5628 gene in A. lentulus with the property of zinc finger protein with KRAB and SCAN domains 3 (http://FungiDB.org, accessed on 10 October 2022); however, its potential functions in growth and AF biosynthesis remain poorly understood.

In this study, the putative C2H2 zinc finger transcription factor encoded by AflZKS3 in A. flavus was characterized, and its intracellular localization and roles in pathogenicity were investigated. Compared with control and complementation strains, AflZKS3 deletion strains showed a reduced growth rate and conidia number, an inability to produce AF, and increased sensitivity to Calcofluor white (CFW) and NaCl stress. The pathogenicity of the deletion mutant was decreased when infecting peanuts and maize. RNA sequencing (RNA-seq) transcriptomic analysis showed that differentially expressed genes (DEGs) in the AflZKS3 deletion strain were mainly associated with growth, oxidative stress, and the biosynthesis of secondary metabolites, including AF and gliotoxin. Our results reveal the potential regulatory mechanism of AflZKS3 in A. flavus growth, cell development, and AF biosynthesis and provide a potential target for controlling A. flavus and AF contamination.

3. DiscussionC2H2 zinc finger transcription factors are known to play vital roles in the development and pathogenicity of microorganisms [16]. In this study, the putative C2H2 zinc finger transcription factor, AflZKS3, annotated in the A. flavus genome, was characterized. The results indicated that this transcription factor is not located in the nucleus and that it plays a major role in the growth and cell development of A. flavus and in AF biosynthesis. Additionally, the potential mechanism was explored by RNA-seq analysis.AflZKS3 was annotated as a putative C2H2 zinc finger transcription factor in the A. flavus genome. The sequence alignment of homologous Aspergillus, Fusarium, and Saccharomyces proteins revealed that AflZKS3 possesses a conserved C2H2 finger domain, implying similar functions. In S. cerevisiae [17] and A. nidulans [18], C2H2 transcription factors are located in the nucleus, but unexpectedly, our results revealed that AflZKS3 was not located in the nucleus. Previous studies have shown that the localization pattern of the iron deficiency-induced transcription factor, bHLH039, in Arabidopsis varies according to the presence of Fer-like iron deficiency-induced transcription factor (FIT) and that bHLH039 is primarily localized in the cytoplasm when expressed in cells lacking FIT, but localized in the nucleus when FIT is present [19]. These results suggested that the subcellular localization of AflZKS3 might be influenced by other regulatory factors resembling bHLH039 in Arabidopsis. We further determined subcellular localization in the presence of CFW, NaCl, and sorbitol and found that AflZKS3 was not located in the nucleus (Figure S3). However, the specific reasons remain to be further explored.C2H2 transcription factors have been shown to play crucial roles in plant and fungal growth and development [20]. Previous studies have reported that the membrane microdomain-associated protein Flotillin 1 (Flot1) is involved in plant growth and development in A. thaliana [21]. In A. fumigatus, the freB gene encoding iron reductase mediates iron metabolism, and the disruption of freB reduces the fungal growth rate, iron reductase activity, and tolerance to oxidative stress [22]. Septins are a conserved GTPase family that play vital roles in growth, meristem, and cell wall integrity. In A. fumigatus, the loss of aspC led to septation, cell wall stress, and meristem defects [23]. Phosphatidylserine decarboxylases (PSDs) are responsible for catalyzing the production of phosphatidylethanolamine, an important phospholipid in homeostasis, growth, and the development of fungi. In A. nidulans, the loss of psdB resulted in severe growth defects, impaired conidia development, and abnormal conidia structure [24]. The present study found that growth-related genes such as FLOT1, freB, aspC, and psd2 were downregulated after the deletion of AflZKS3, indicating that iron metabolism, GTPases, and phospholipid homeostasis might be regulated by AflZKS3, and thereby affect mycelia growth. Additionally, con-6, a conidia-related gene, is relatively conserved in filamentous fungi and preferentially expressed during conidia development [25]. The vosA gene encodes a key regulator of Aspergillus spores and is essential for the morphological development and metabolic integrity of conidia. Previous studies found that a vosA mutant strain displayed defective growth on media supplemented with Congo red, sodium chloride, and sorbitol [26]. Herein, we found that genes associated with spore development in the strain ΔAflZKS3, such as con-6, vosA, cetA, betA, AQY1, spore wall-specific gene DIT2 [27], and conidia lipid homeostasis-related gene SAY1 [28] were all downregulated. Furthermore, previous studies indicated that CFW is specifically bound to chitin, while CR is bound to β-1, 3-glucan, thus obstructing the normal assembly of the cell wall, resulting in cell wall stress, and inhibiting the growth of the cell [29]. Our results demonstrated that the AflZKS3 deletion strain showed different sensitivity to CFW and CR compared with A. flavus control and the AflZKS3-Com strains, which might be attributed to their different mechanisms of action and the cell wall defects caused by AflZKS3 deletion. Additionally, the AflZKS3 deletion strain was much more sensitive to NaCl than sorbitol compared with the control and AflZKS3-Com strains. The possible reason might be that NaCl belongs to the category of ionic and cell penetrating agent, which can induce ionic stress and produce specific ionic toxicity, while sorbitol belongs to non-ionic osmotic stress agent. Previous research also demonstrated that the induced effect of NaCl is more profound than that of sorbitol in Japonica rice [30,31].The cell wall of fungi is a complex structure composed mainly of chitin and glucan and plays a vital role in morphogenesis and protection from various environmental stresses [32]. ChiA is a class III chitinase involved in spore germination and mycelial growth [33]. Agn1, which encodes 1, 3-α-glucanase, is involved in cell division [34]. Gel2 and glx3 are associated with cell wall integrity [35,36]. These results indicate that AflZKS3 might affect fungal morphogenesis, defense responses, and cell division by downregulating cell wall-related genes chiA, agn1, gel2, gel4, glx3, and gpi13. We found that the AFLA_02641 deletion mutant displayed increased sensitivity to CFW, similar to the glx3 deletion strain in Candida albicans [36].A. flavus growth has been reported to be closely related to AF biosynthesis [37]. Impaired growth and conidia development are often accompanied by secondary metabolism disruption. The biosynthesis of AFs is a complex enzymatic process involving 21 enzymes encoded by a gene cluster ~70 kb in size [38]. Studies have shown that the biosynthesis of fatty acids is involved in the initial stage of biosynthesis, fatty acid synthase is involved in the formation of polyketide initiation units of AFs, and high fatty acid synthase activity can promote AFB1 production [39]. Furthermore, fas-1, which encodes fatty acid synthase, is required for the biosynthesis of norsolorinic acid and AFs [40]. AflQ encodes an oxidoreductase involved in the formation of the AFB1 precursor hydroxyl-methylsterigmatocystin, and it plays a role in the latter stages of the biosynthetic pathway [41]. There is a strong linear relationship between aflQ expression and the AF-producing capacity of A. flavus and A. parasiticus [42]. In this study, the deletion of AflZKS3 downregulated the AF biosynthesis-related genes, fasA, aflQ, aflB, and aflF. O-methyltransferase, another key enzyme in AFB1 synthesis, catalyzes the transformation of sterigmatocystin to O-methylsterigmatocystin and dihydrosterigmatocystin to dihydro-O-methylsterigmatocystin [38]. Cytochrome P450 enzymes are involved in the formation of sterigmatocystin, a late intermediate in the AFB1 synthesis pathway [43]. We found that genes associated with O-methyltransferase (imqG, aclH) and cytochrome P450 (lnaC, BOT4) were downregulated in AflZKS3 mutants. These results indicate that AFB1 production can be inhibited by AflZKS3 through the regulation of multiple genes involved in AF biosynthesis.In addition to AF biosynthesis, genes involved in other secondary metabolic pathways were also affected. Gliotoxin is synthesized by a biosynthetic gene cluster of 12 genes in A. fumigatus [44]. GliA is involved in gliotoxin biosynthesis and has important functions in gliotoxin export and fungal self-protection. It was found that the disruption of gliA greatly reduced the production of gliotoxin [45]. We found that the gliA gene related to gliotoxin biosynthesis was downregulated. Additionally, polyketide synthases and non-ribosomal peptide synthetases are large multimodular enzymes that participate in the biosynthesis of polyketides and peptide secondary metabolites [46]. Among them, polyketides are the most abundant fungal secondary metabolites, and they are synthesized by a type I diketone synthase [43]. Previous studies have revealed that the deletion of the pksCT gene in Monascus decreased citrinin production capacity by >98% [47]. In this study, genes associated with polyketide synthase and non-ribosomal peptide synthase (albA, nscA, pksCT, and lnaA) were also downregulated. These results suggest that AflZKS3 might play a global regulatory role in mycotoxin export and the self-protection of fungi.Oxidative stress in filamentous fungi is often associated with secondary metabolism, and it is also one of the prerequisites for AF production. Studies have found that low concentrations of reactive oxygen species (ROS) can stimulate the synthesis of secondary metabolites; conversely, high concentrations of ROS are toxic to cells, even causing cell death, and they are detrimental to the biosynthesis of secondary metabolites [48]. A variety of antioxidant enzymes produced by cells, such as superoxide dismutase, peroxidase, and catalase, remove excess ROS to protect cells from oxidative stress [49]. Previous studies have indicated that oxr1 encodes an antioxidant regulator that protects against intracellular H2O2-induced oxidative stress [50]. SsuD encodes an alkane sulfonate monooxygenase that also protects cells from oxidative stress [51]. In our current study, transcriptome data showed that the deletion of AflZKS3 downregulated antioxidant-related genes sodB, cat1, oxr1, and ssuD, and salt stress-related genes dur3 and phoD [52,53], which might be responsible for the observed changes in AFB1 and other secondary metabolites.

In conclusion, we investigated the putative C2H2 zinc finger transcription factor AflZKS3 in A. flavus, and the results indicated that deletion of AflZKS3 inhibited cell growth, conidia formation, and AFB1 biosynthesis ability. RNA-seq was used to further investigate its underlying regulatory mechanism, and the analysis of DEGs indicated that growth-related genes (FLOT1, psd2, vosA, con-6, and gel2), secondary metabolism-related genes (aflB, aflF, aflQ, and pksCT), and oxygen stress-related genes (sodB, cat1, and oxr1) were downregulated. Therefore, the putative C2H2 zinc finger transcription factor AflZKS3 regulates growth, cell development, and oxidative stress-related genes, and affects the secondary metabolism in A. flavus. These results further our understanding of the functions of C2H2 zinc finger transcription factors in fungal pathogenicity regulation and provide a potential target for developing novel control strategies in A. flavus.

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