Aflatoxin Biosynthesis Research Articles

Quantitative Proteomic Analysis for High- and Low-Aflatoxin-Yield Aspergillus flavus Strains Isolated From Natural Environments.

The molecular mechanisms underlying aflatoxin production have been well-studied in strains of the fungus Aspergillus flavus (A. flavus) under artificial conditions. However, aflatoxin biosynthesis has rarely been studied in A. flavus strains isolated from field conditions with different aflatoxin-producing ability. In the present study, tandem mass tag (TMT) labeling and high-performance liquid chromatography (HPLC) coupled with tandem-mass spectrometry analysis were used for proteomic quantification in natural isolates of high- and low-aflatoxin-yield A. flavus strains. Additionally, findings obtained using the TMT-labeling method were validated using the high-resolution multiple reaction monitoring (MRM-HR) method. In total, 4,363 proteins were quantified, among which 1,045 proteins were differentially expressed between the high- and low-aflatoxin-yield A. flavus strains. Bioinformatics analysis showed that the up-regulated proteins were significantly enriched in carbon-related metabolism and the biosynthesis of secondary metabolites, whereas the down-regulated proteins were enriched in oxidative phosphorylation. Moreover, GST proteins were found to be significantly down-regulated in high-yield A. flavus strains; this result contradicted previous findings obtained from A. flavus strains grown under artificial conditions. In summary, our study provides novel insights into aflatoxin regulation in A. flavus under field conditions and could facilitate the development of various strategies for the effective control of aflatoxin contamination in food crops.

Antifungal properties and mechanisms of three volatile aldehydes (octanal, nonanal and decanal) on Aspergillus flavus

The harm of Aspergillus flavus ( A. flavus ) and aflatoxin is a severe food safety problem worldwide, which causes huge economic losses every year. Therefore, it is urgent to control the growth of A. flavus and the biosynthesis of aflatoxin. Plant-derived natural compounds are superior to synthetic fungicide in inhibiting the growth of A. flavus benefiting from their high safety to the environment, humans and stock, and low cost. This study aimed to evaluate the antifungal effects and potential antifungal mechanisms of three plant-derived compounds (octanal, nonanal and decanal) against A. flavus . We determined the minimum inhibitory concentrations (MICs) and action mechanism of the three volatile aldehydes on A. flavus and also performed calcofluor white (CW) staining for visualizing the distribution of septa. Cell respiration metabolism and the pathogenicity on maize kernels were also carried out to evaluate the efficacy of the three volatile aldehydes on the growth of A. flavus . The results showed that the three volatile aldehydes could inhibit the germination of spores and mycelial growth of A. flavus , the MICs on spores and mycelia were: octanal (1.0 and 0.5 μL/mL), nonanal (0.5 and 2.0 μL/mL), and decanal (1 and 5 μL/mL). The three volatile aldehydes could strongly damage the integrity of both the cell wall and the cell membrane of A. flavus . Meanwhile, they could decrease the content of total lipid and inhibit respiration metabolism of A. flavus cell. Results of in vitro antifungal test showed that all the three volatile aldehydes could effectively prevent the growth of A. flavus on maize kernels. The study revealed that octanal, nonanal and decanal could effectively inhibit the growth of A. flavus both in culture medium and on maize kernels to different extent. The results confirmed that the plant-derived compounds could be developed into promising antifungal agents applied in the preservation of grains. This study provides a theoretical basis for the research and application of potential antifungal agents.

Down-regulation of aflatoxin biosynthetic genes in Aspergillus parasiticus by Heliopsis longipes roots and affinin for reduction of aflatoxin production

Affinin present in Heliopsis longipes roots has been identified as an anti-aflatoxin molecule. However, its mechanism of action has yet to be clarified. Aflatoxins biosynthesis involves not less than 27 enzymatic reactions. In this work, the genes aflG, aflH, aflI, aflK, aflL, aflM, aflO, aflP, and aflQ of the aflatoxins cluster and the aflS gene encoding an internal regulatory factor involved in aflatoxins biosynthesis in Aspergillus parasiticus, were studied by qRT-PCR. Results demonstrated that ethanolic extract of H. longipes roots and affinin inhibit aflatoxin biosynthesis and fungal growth in a dose-dependent manner. At 300 µg/mL, ethanolic extract and affinin presented the highest inhibition of radial growth (86% and 94%) and aflatoxin production (68% and 80%). The qRT-PCR analysis demonstrated that nine tested genes were down-regulated by affinin and ethanolic extract. The most down-regulated was the aflK, a gene that encodes an enzyme cyclase with double function during the aflatoxin biosynthesis. While no significant down-regulation was obtaining for aflH gene. Exposure to affinin also resulted in decreased transcript levels of the internal regulator factor aflS. Based on our results, a model showing the regulatory mechanism in aflatoxin biosynthesis and its role in gene expression was proposed. In conclusion, affinin modulates the expression of several aflatoxin biosynthetic genes, leading to mycotoxin biosynthesis inhibition. Therefore, H. longipes roots is a suitable candidate to developed control strategies via lowering gene expressions as a future perspective in reducing aflatoxin contamination.

Updates on the Functions and Molecular Mechanisms of the Genes Involved in Aspergillus flavus Development and Biosynthesis of Aflatoxins.

Aspergillus flavus (A. flavus) is a ubiquitous and opportunistic fungal pathogen that causes invasive and non-invasive aspergillosis in humans and animals. This fungus is also capable of infecting a large number of agriculture crops (e.g., peanuts, maze, cotton seeds, rice, etc.), causing economic losses and posing serious food-safety concerns when these crops are contaminated with aflatoxins, the most potent naturally occurring carcinogens. In particular, A. flavus and aflatoxins are intensely studied, and they continue to receive considerable attention due to their detrimental effects on humans, animals, and crops. Although several studies have been published focusing on the biosynthesis of the aforementioned secondary metabolites, some of the molecular mechanisms (e.g., posttranslational modifications, transcription factors, transcriptome, proteomics, metabolomics and transcriptome, etc.) involved in the fungal development and aflatoxin biosynthesis in A. flavus are still not fully understood. In this study, a review of the recently published studies on the function of the genes and the molecular mechanisms involved in development of A. flavus and the production of its secondary metabolites is presented. It is hoped that the information provided in this review will help readers to develop effective strategies to reduce A. flavus infection and aflatoxin production.

Licorice, Doum, and Banana Peel Extracts Inhibit Aspergillus flavus Growth and Suppress Metabolic Pathway of Aflatoxin B1 Production

Three different concentrations of four (ethanol, acetone, methanol, and diethyl ether) extracts of licorice, doum, and banana peel were evaluated for antifungal and antimycotoxigenic efficiency against a maize aflatoxigenic fungus, Aspergillus flavus. Among them, the licorice diethyl ether 75% extract was intensely active, showing the best wet and dry weight inhibition and exhibiting the highest efficacy ratio (91%). Regarding aflatoxin B1 (AFB1) production, all the plant extracts tested were effective against AFB1 production after one month of maize storage, with average efficacy ratios ranging from 74.1% to 97.5%. At the same time, Thiram fungicide exhibited an efficacy ratio of 20.14%. The relative expression levels of three structural genes (aflD, aflP, and aflQ) and two regulatory genes (aflR and aflS) were significantly downregulated when compared to untreated maize grains or Thiram-treated maize grains. The doum diethyl ether 75% peel extract showed the highest total phenolic content (60.48 mg GAE/g dry extract wt.) and antioxidant activity (84.71 μg/mL). GC–MS analysis revealed that dimethoxycinnamic acid, aspartic acid, valproic acid, and linoleic acid might imbue the extracts with antioxidant capacities in relation to fungal growth and aflatoxin biosynthesis. Finally, the results suggest that the three plant extracts can be considered a promising source for developing potentially effective and environmentally safer alternative ways to control aflatoxin formation, thus creating a potentially protective method for grain storage.

Aflatoxin Biosynthesis, Genetic Regulation, Toxicity, and Control Strategies: A Review.

Aflatoxins (AFs) are highly toxic and cancer-causing compounds, predominantly synthesized by the Aspergillus species. AFs biosynthesis is a lengthy process that requires as minimum as 30 genes grouped inside 75 kilobytes (kB) of gene clusters, which are regulated by specific transcription factors, including aflR, aflS, and some general transcription factors. This paper summarizes the status of research on characterizing structural and regulatory genes associated with AF production and their roles in aflatoxigenic fungi, particularly Aspergillus flavus and A. parasiticus, and enhances the current understanding of AFs that adversely affect humans and animals with a great emphasis on toxicity and preventive methods.

The Regulatory Mechanism of Water Activities on Aflatoxins Biosynthesis and Conidia Development, and Transcription Factor AtfB Is Involved in This Regulation.

Peanuts are frequently infected by Aspergillus strains and then contaminated by aflatoxins (AF), which brings out economic losses and health risks. AF production is affected by diverse environmental factors, especially water activity (aw). In this study, A. flavus was inoculated into peanuts with different aw (0.90, 0.95, and 0.99). Both AFB1 yield and conidia production showed the highest level in aw 0.90 treatment. Transcriptional level analyses indicated that AF biosynthesis genes, especially the middle- and later-stage genes, were significantly up-regulated in aw 0.90 than aw 0.95 and 0.99. AtfB could be the pivotal regulator response to aw variations, and could further regulate downstream genes, especially AF biosynthesis genes. The expressions of conidia genes and relevant regulators were also more up-regulated at aw 0.90 than aw 0.95 and 0.99, suggesting that the relative lower aw could increase A. flavus conidia development. Furthermore, transcription factors involved in sexual development and nitrogen metabolism were also modulated by different aw. This research partly clarified the regulatory mechanism of aw on AF biosynthesis and A. flavus development and it would supply some advice for AF prevention in food storage.

Ras subfamily GTPases regulate development, aflatoxin biosynthesis and pathogenicity in the fungus Aspergillus flavus.

Ras subfamily proteins are molecular switches in signal transduction pathways of many eukaryotes that regulate a variety of cellular processes. Here, the Ras subfamily, encoded by six genes, was identified in Aspergillus flavus: rasA, rasB, rasC, rab-33, rheb and rsr1. The rsr1 deletion mutant (∆rsr1), rheb deletion mutant (∆rheb) and double deletion mutant (∆rheb/rsr1) displayed significantly decreased growth and sporulation. Sclerotia formation was significantly decreased for ∆rheb or ∆rheb/rsr1 but increased for ∆rsr1. Aflatoxin production was significantly increased in ∆rheb but decreased in ∆rsr1 and ∆rheb/rsr1. We found that rsr1 and rheb are crucial for the pathogenicity of A. flavus. Quantitative proteomics identified 520 differentially expressed proteins (DEPs) for the ∆rsr1 mutant and 133 DEPs for the ∆rheb mutant. These DEPs were annotated in multiple biological processes and KEGG pathways in A. flavus. Importantly, we identified the cytokinesis protein SepA in the protein-protein interaction network of rsr1, and deletion mutants showed that SepA has pleiotropic effects on growth and AF biosynthesis, which may depend on Rsr1 for regulation in A. flavus. Our results indicated that these Ras subfamily proteins exhibited functional redundancy with each other but there were also differences in A. flavus.

Quantification of the Aflatoxin Biocontrol Strain Aspergillus flavus AF36 in Soil and in Nuts and Leaves of Pistachio by Real-Time PCR.

The species Aspergillus flavus and A. parasiticus are commonly found in the soils of nut-growing areas in California. Several isolates can produce aflatoxins that occasionally contaminate nut kernels, conditioning their sale. Strain AF36 of A. flavus, which does not produce aflatoxins, is registered as a biocontrol agent for use in almond, pistachio, and fig crops in California. After application in orchards, AF36 displaces aflatoxin-producing Aspergillus spp. and thus reduces aflatoxin contamination. Vegetative compatibility assays (VCAs) have traditionally been used to track AF36 in soils and crops where it has been applied. However, VCAs are labor intensive and time consuming. Here, we developed a quantitative real-time PCR (qPCR) protocol to quantify proportions of AF36 accurately and efficiently in different substrates. Specific primers to target AF36 and toxigenic strains of A. flavus and A. parasiticus were designed based on the sequence of aflC, a gene essential for aflatoxin biosynthesis. Standard curves were generated to calculate proportions of AF36 based on threshold cycle values. Verification assays using pure DNA and conidial suspension mixtures demonstrated a significant relationship by regression analysis between known and qPCR-measured AF36 proportions in DNA (R2 = 0.974; P < 0.001) and conidia mixtures (R2 = 0.950; P < 0.001). Tests conducted by qPCR in pistachio leaves, nuts, and soil samples demonstrated the usefulness of the qPCR method to precisely quantify proportions of AF36 in diverse substrates, ensuring important time and cost savings. The outputs of this study will serve to design better aflatoxin management strategies for pistachio and other crops.

Diversity and composition of the North Sikkim hot spring mycobiome using a culture-independent method.

Fungi are considered to be the most resilient and economically important microbial community that can easily survive and optimally grow under a wide range of growth conditions. Thermophilic fungi from the geothermal sources have been less pondered upon and lie unexplored. Here, a microbiome approach was conducted to understand the concealed world of the environmental mycobiota from the two hot springs of North Sikkim district located in North-east India. The solfataric muds from the hot springs were analyzed. In both the samples, on the basis of genus level classification, genus Fusarium had the highest abundance followed by Colletotrichum, Pochonia, Pyricularia, Neurospora, etc. Analyzing the predicted genes, the functional proteins of New Yume Samdung mycobiome were found to be dominated by the genera Fusarium (22%), Trichoderma (12%), and Aspergillus (11%), whereas in the case of Old Yume Samdung, it was dominated by the genera Aspergillus (11%), Saccharomyces (6%), and Fusarium (5%). Interestingly, in the studied mycobiome, environmental yeasts were also detected. From the functional metagenomics, sulfate adenylatetransferase (SAT) proteins for sulfur assimilation were found in some of the fungal reads. Toxin protein reads such as AM-toxin biosynthesis proteins, AF-toxin biosynthesis proteins, Gliotoxin biosynthesis proteins, and aflatoxin biosynthesis proteins were detected in the mycobiomes.

In silico efficacy of [S]-8-gingerol (a derivative) with 6-gingerol against PT-domain of Polyketide synthase A (PksA)

The PT-domain of Polyketide synthase A (PksA) is crucial product template that controls the aldol cyclization and aromatization to create polyketide precursor. The precursor leads to biosynthesis of aflatoxin secondary metabolites. Being carcinogenic in nature of these secondary metabolic, efforts have been made to explore novel inhibitors targeting the PT-domain of PksA. Previously, [6]-gingerol reported as a prospective inhibitor for the inhibition for aflatoxin biosynthesis, but some of its derivatives such as (S)- [8]-gingerol, 9-Hydroxy-[6]-gingerol, 9-gingerol, 5-O-methyl-[9]-gingerol, methyl-6-gingerol, and [5]-gingerol exhibited better binding affinity. Notably, Leu1508, Asn1554, and Asn1568, are the key amino acid residues that are involved in stabilizing ligand-protein interaction. The study explores the understanding to develop new approach to create novel drug against the PT-domain to restrict the aflatoxin biosynthesis at initiation. However, a detailed in vivo study is required to further evaluation at in vivo stage.

Molecular analysis of aflR gene in Aspergillus flavus isolated from Iran

Aspergillus flavus produces the most potent carcinogens, aflatoxins, when it contaminates agricultural crops. aflR gene regulates aflatoxin-related genes and it has been identified in four species of A. flavus, A. parasiticus, A. sojae and A. oryzae. Contamination of agricultural commodities with aflatoxin is a grave risk to humans and animals’ health. Aflatoxin related genes are clustered in a 75 kb region of genome in A. flavus. Investigations obviously demonstrated that aflatoxin biosynthesis needs the aflR gene product and an entirely functional aflatoxin biosynthetic cluster. The purpose of the current study was to investigate the presence of the aflR gene in A. flavus. Material and methods. Forty-two A. flavus isolates including 10 references, 25 clinical and 7 environmental isolates were analyzed in this study. The isolates were identified by morphology. To characterize morphologically, the conidial arrangement, philiades, vesicles and conidiophores were observed microscopically. Using PCR, the aflR gene was amplified with primers aflR1 and aflR2. PCR were carried out to amplify an 800 bp DNA fragment of aflR gene. Some amplicons were sequenced. The sequences were searched in NCBI database and analyzed with MEGA5 software. Results and discussion. Out of 42 A. flavus isolates, an 800 bp band was amplified for 35 isolates. No band was observed for seven isolates including 4 clinical and 3 environmental isolates. Data analysis demonstrated that 100% of reference strains and 84% of clinical strains produced the expected fragment while it was only 57.14% for environmental isolates. The sequences had 100% identity with A. flavus aflR gene which was deposited in the NCBI database. Conclusion. In conclusion, molecular analysis of the aflR gene showed that this gene was not amplified from some strains of A.flavus; therefore, perhaps it lacks the gene or it is greatly abnormal. Additional researches are needed to verify whether the strains with lack of aflR gene have a loss of function in production of aflatoxin or other mechanisms of regulation exist

Transcriptomic responses of Aspergillus flavus to temperature and oxidative stresses during aflatoxin production

Aflatoxin is a group of polyketide-derived carcinogenic and mutagenic secondary metabolites produced by Aspergillus flavus that negatively impact global food security and threaten the health of both humans and livestock. Aflatoxin biosynthesis is strongly affected by the fungal developmental stage, cultivation conditions, and environmental stress. In this study, a novel float culture method was used to examine the direct responses of the A. flavus transcriptome to temperature stress, oxidative stress, and their dual effects during the aflatoxin production stage. The transcriptomic response of A. flavus illustrated that the co-regulation of different secondary metabolic pathways likely contributes to maintaining cellular homeostasis and promoting cell survival under stress conditions. In particular, aflatoxin biosynthetic gene expression was downregulated, while genes encoding secondary metabolites with antioxidant properties, such as kojic acid and imizoquins, were upregulated under stress conditions. Multiple mitochondrial function-related genes, including those encoding NADH:ubiquinone oxidoreductase, ubiquinol-cytochrome C reductase, and alternative oxidase, were differentially expressed. These data can provide insights into the important mechanisms through which secondary metabolism in A. flavus is co-regulated and facilitate the deployment of various approaches for the effective control and prevention of aflatoxin contamination in food crops.

Proteogenomic Characterization of the Pathogenic Fungus Aspergillus flavus Reveals Novel Genes Involved in Aflatoxin Production

AbstractAspergillus flavus (A. flavus), a pathogenic fungus, can produce carcinogenic and toxic aflatoxins that are a serious agricultural and medical threat worldwide. Attempts to decipher the aflatoxin biosynthetic pathway have been hampered by the lack of a high-quality genome annotation for A. flavus. To address this gap, we performed a comprehensive proteogenomic analysis using high-accuracy mass spectrometry data for this pathogen. The resulting high-quality data set confirmed the translation of 8724 previously predicted genes and identified 732 novel proteins, 269 splice variants, 447 single amino acid variants, 188 revised genes. A subset of novel proteins was experimentally validated by RT-PCR and synthetic peptides. Further functional annotation suggested that a number of the identified novel proteins may play roles in aflatoxin biosynthesis and stress responses in A. flavus. This comprehensive strategy also identified a wide range of posttranslational modifications (PTMs), including 3461 modification sites from 1765 proteins. Functional analysis suggested the involvement of these modified proteins in the regulation of cellular metabolic and aflatoxin biosynthetic pathways. Together, we provided a high-quality annotation of A. flavus genome and revealed novel insights into the mechanisms of aflatoxin production and pathogenicity in this pathogen.

The inhibitory mechanism of methyl jasmonate on Aspergillus flavus growth and aflatoxin biosynthesis and two novel transcription factors are involved in this action

As the most toxic and carcinogenic mycotoxin, aflatoxin B1 (AFB1) biosynthesis depends on a series of enzymatic reactions and a complicated regulatory system. Methyl jasmonate (MeJA) is one of stress associated phytohormones. In this study, MeJA could inhibit A. flavus growth and AFB1 production with a dose-dependent manner. SEM and TEM analysis indicated that morphological ultrastructure deteriorations were observed in A. flavus treated with MeJA. RNA-Seq indicated that the initial-steps aflatoxins (AFs) genes were no drastic difference, but the middle- and later- steps genes were significantly down-regulated, which might be due to the decreases of global regulators, especially AtfB. More importantly, two novel regulators (AFLA_085880 and AFLA_015850) were involved in the inhibition, and were recognized as the critically positive regulators for AFs productions. The two genes mutants also showed significantly decrease expressions of AFs cluster genes and AFs associated regulators, and subsequent AFB1 biosynthesis. This research partly clarified inhibitory mechanism of MeJA and made some contributions to the elimination of AFs contamination.

A novel phosphoinositide kinase Fab1 regulates biosynthesis of pathogenic aflatoxin in Aspergillus flavus

ABSTRACT Aspergillus flavus (A. flavus) is one of the most important model environmental fungi which can produce a potent toxin and carcinogen known as aflatoxin. Aflatoxin contamination causes massive agricultural economic loss and a critical human health issue each year. Although a functional vacuole has been highlighted for its fundamental importance in fungal virulence, the molecular mechanisms of the vacuole in regulating the virulence of A. flavus remain largely unknown. Here, we identified a novel vacuole-related protein in A. flavus, the ortholog of phosphatidylinositol-3-phosphate-5-kinase (Fab1) in Saccharomyces cerevisiae. This kinase was located at the vacuolar membrane, and loss of fab1 function was found to affect the growth, conidia and sclerotial development, cellular acidification and metal ion homeostasis, aflatoxin production and pathogenicity of A. flavus. Further functional analysis revealed that Fab1 was required to maintain the vacuole size and cell morphology. Additional quantitative proteomic analysis suggested that Fab1 was likely to play an important role in maintaining vacuolar/cellular homeostasis, with vacuolar dysregulation upon fab1 deletion leading to impaired aflatoxin synthesis in this fungus. Together, these results provide insight into the molecular mechanisms by which this pathogen produces aflatoxin and mediates its pathogenicity, and may facilitate dissection of the vacuole-mediated regulatory network in A. flavus.

The Conserved MAP Kinase MpkB Regulates Development and Sporulation without Affecting Aflatoxin Biosynthesis in Aspergillus flavus.

In eukaryotes, the MAP kinase signaling pathway plays pivotal roles in regulating the expression of genes required for growth, development, and stress response. Here, we deleted the mpkB gene (AFLA_034170), an ortholog of the Saccharomyces cerevisiae FUS3 gene, to characterize its function in Aspergillus flavus, a cosmopolitan, pathogenic, and aflatoxin-producing fungus. Previous studies revealed that MpkB positively regulates sexual and asexual differentiation in Aspergillus nidulans. In A. flavus, mpkB deletion resulted in an approximately 60% reduction in conidia production compared to the wild type without mycelial growth defects. Moreover, the mutant produced immature and abnormal conidiophores exhibiting vesicular dome-immaturity in the conidiophore head, decreased phialide numbers, and very short stalks. Interestingly, the ΔmpkB mutant could not produce sclerotia but produced aflatoxin B1 normally. Taken together, these results suggest that the A. flavus MpkB MAP kinase positively regulates conidiation and sclerotia formation but is not involved in the production of secondary metabolites such as aflatoxin B1.

Ssu72 Regulates Fungal Development, Aflatoxin Biosynthesis and Pathogenicity in Aspergillus flavus

The RNA polymerase II (Pol II) transcription process is coordinated by the reversible phosphorylation of its largest subunit-carboxy terminal domain (CTD). Ssu72 is identified as a CTD phosphatase with specificity for phosphorylation of Ser5 and Ser7 and plays critical roles in regulation of transcription cycle in eukaryotes. However, the biofunction of Ssu72 is still unknown in Aspergillus flavus, which is a plant pathogenic fungus and produces one of the most toxic mycotoxins-aflatoxin. Here, we identified a putative phosphatase Ssu72 and investigated the function of Ssu72 in A. flavus. Deletion of ssu72 resulted in severe defects in vegetative growth, conidiation and sclerotia formation. Additionally, we found that phosphatase Ssu72 positively regulates aflatoxin production through regulating expression of aflatoxin biosynthesis cluster genes. Notably, seeds infection assays indicated that phosphatase Ssu72 is crucial for pathogenicity of A. flavus. Furthermore, the Δssu72 mutant exhibited more sensitivity to osmotic and oxidative stresses. Taken together, our study suggests that the putative phosphatase Ssu72 is involved in fungal development, aflatoxin production and pathogenicity in A. flavus, and may provide a novel strategy to prevent the contamination of this pathogenic fungus.

Deleterious missense variants in the aflatoxin biosynthesis genes explain the low toxicity of Aspergillus flavus from infected rice

Aspergillus flavus is one of the most natural contaminants of the improperly stored rice grains. It produces several secondary metabolites, like aflatoxins, which are well known hepatotoxic, hepatocarcinogenic and mutagenic agents. This study describes the in silico consequences of the missense mutations identified in several genes of aflatoxins biosynthesis in rice-contaminating A. flavus isolates. In the in vitro portion of the study, aflatoxins production profile was measured, and PCR-single strand-conformation polymorphism (SSCP)-sequencing method was used to genotype the studied genetic loci: aflP, aflM, aflR, PEP, and cob. Results showed aflatoxigenic potential in 79 out of 109 A. flavus isolates. Twenty-two missense and fifty-five synonymous mutations were found to be distributed variably on the studied loci. In the in silico portion of this study, several computations were utilized to predict the effect of each observed missense mutation on proteins structure, function, and stability. Seven mutations (O-methyl transferase: p.G256C; ver-1 dehydrogenase: p.K179 N and p.V183L; aspergillopepsin-1: p.P137L, p.S138F, p.G154C, and p.S158C) were found to be highly deleterious among the missense variants with damaging effects on their proteins’ structure and function. In contrast to these detected variations in the aflatoxigenic loci, all missense mutations in the control non-aflatoxigenic cob gene were found to be neutral. These findings indicated that the observed mutations may reduce the concomitant biohazard of their biosynthesized aflatoxins. The current findings suggest that the naturally available variants may reduce or eliminates the dangerous consequences of aflatoxins upon ingesting the rice infected with A. flavus. To the best of our knowledge, this study is the first comprehensive report to analyze the missense mutations on the aflatoxin biosynthesis genes using in vitro and the state-of-art bio-computational tools.

The membrane mucin Msb2 regulates aflatoxin biosynthesis and pathogenicity in fungus Aspergillus flavus.

As a pathogenic fungus, Aspergillus flavus can produce carcinogenic aflatoxins (AFs), which poses a great threat to crops and animals. Msb2, the signalling mucin protein, is a part of mitogen-activated protein kinase (MAPK) pathway which contributes to a range of physiological processes. In this study, the roles of membrane mucin Msb2 were explored in A. flavus by the application of gene disruption. The deletion of msb2 gene (Δmsb2) caused defects in vegetative growth, sporulation and sclerotia formation when compared to WT and complement strain (Δmsb2C ) in A. flavus. Using thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analysis, it was found that deletion of msb2 down-regulated aflatoxin B1 (AFB1 ) synthesis and decreased the infection capacity of A. flavus. Consistently, Msb2 responds to cell wall stress and osmotic stress by positively regulating the phosphorylation of MAP kinase. Notably, Δmsb2 mutant exhibited cell wall defect, and it was more sensitive to inhibitor caspofungin when compared to WT and Δmsb2C . Taking together, these results revealed that Msb2 plays key roles in morphological development process, stresses adaptation, secondary metabolism and pathogenicity in fungus A. flavus.