| Aspergillus flavus(A.flavus)is one of the most important species in the Aspergillus genus that can cause both non-invasive and invasive systematic aspergillosis in immunocompromised individuals,animals and economically-important crops.A.flavus is capable of producing a range of potent carcinogens and toxins collectively referred to as aflatoxins,which represent a significant concern due to the potential for environmental contamination with these compounds.When foods contaminated with aflatoxin are ingested,or when A.flavus is able to grow without immunological constraint,this can lead to acute toxicity and a long-term increase in cancer risk in affected individuals.Therefore,the study of the genes involved in aflatoxin biosynthesis will enable us to understand the molecular mechanisms of aflatoxin biosynthesis and pathway regulation and control the A.flavus and aflatoxins.Although the genome annotation of A.flavus has been completed and the related toxin-producing gene clusters have been revealed,the aflatoxin synthesis,transportation and secretion have not been clarified and the genes in many key pathways have not been clarified.The present study was designed to conduct the high-quality comprehensive annotation of the A.flavus genome through an integrated proteogenomic approach in order to identify novel proteins and to explore the functional role of a novel hosphoinositide kinase(Fab1)encoded by this pathogenic microbe.In this study,we are aware of to have employed such a proteogenomcis approach to improve Aspergillus genomic annotation.By using a stringent filtering threshold(FDR < 1%),we confirmed the translation of 8,724 previously-annotated protein-coding genes,indicating the presence of these genes at protein level.However,the remaining predicted genes were still not detected by any peptide evidence in this analysis,leading us to next determine whether they correspond to non-coding genes in the A.flavus database,based upon bioinformatics analysis.We inferred that 3,279 genes were not protein coding-genes in the A.flavus database.In addition,the proteogenomics datasets resulted in the identification of 732 novel protein-coding genes,188 revised genes,543 alternatively spliced(AS)genes and 447 single amino acid variants(SAAVs).Functional annotation revealed that most of these evolutionary conserved novel proteins were associated with multiple cellular processes,including stress responses and aflatoxin biosynthesis.These results showed that there is a pressing need for a high-quality annotation of protein coding genes for A.flavus,owing to the presence of incorrectly annotated protein-coding genes and undiscovered protein-coding genes.Our findings could provide important resources and new research directions for the study of the mechanism of aflatoxin production in A.flavus.The in-deep proteogenomics data also enabled us to systematicall alyze the post-translational modification(PTM)of proteins in A.flavus.We next conducted a global systematic investigation of the most frequently detected PTMs in A.flavus based on open-search protein modification search approach.By using these strategies,a total of 6,147 proteins with different PTMs were identified in A.flavus.According to the restricted protein modification search strategy,our approach identified 24 different PTMs,indicating that there existed complicated PTM events.The in-depth identification of more than 20 PTMs also enabled us to analyze the function of these modified proteins,revealing these modified proteins to play diverse roles as regulators of many cellular processes,including metabolic pathways,biosynthesis of aflatoxin and other secondary metabolites.Our western-blot analysis further suggested that the identified PTMs could play an important role in many cellular processes.Based on the preteogenomics analysis and functional annotation,a novel phosphoinositide kinase(Fab1)was identified and we then studied its bio-functions in A.flavus growth,aflatoxin production and pathogenicity.Our results revealed that loss of fab1 function affected the growth,conidia and sclerotial development,cellular acidification and metal ion homeostasis,aflatoxin production and pathogenicity of A.flavus.We additionally assayed the expression of the related genes and found that the related genes in aflatoxin biosynthesis were significantly downregulated.In order to assess Fab1 subcellular ocalization in A.flavus,we first produced a Fab1-GFP fusion protein and we observed robust Fab1 localization to the vacuolar membrane.Notably,we observed marked increases in vacuole size in ?fab1 fungi,with vacuoles having swollen to fill most of the cell volume.Moreover,we isolated the vesicle-vacuole fraction from A.flavus cells and our results suggested that aflatoxins biosynthesis and transport may be disrupted in the ?fab1 strain.We speculated that Fab1 was required for maintenance of vacuole size and cell morphology.To further examine the molecular basis for Fab1 functionality,we performed a TMT-based quantitative proteomic analysis to identify proteins regulated by Fab1 in A.flavus.Finally,we quantified 3,821 proteins and identified 289 differently expressed proteins(DEPs).Among these,163 were up-regulated and 126 were down-regulated.Further functional annotation revealed that a large portion of these DEPs were involved in metabolic processes,biosynthesis,transport and stress-response activities.The majority of these DEPs were located in vacuole,endoplasmic reticulum,Golgi,peroxisomes,membrane,extracellular and cytosol.Pathway analysis indicated that a subset of DEPs to be involved in growth,development,aflatoxin biosynthesis,transport and secretion in A.flavus.These analysis suggested that Fab1 was likely to play an important role in maintaining vacuolar/cellular homeostasis,with vacuolar dysregulation upon fab1 deletion leading to mpaired aflatoxin synthesis in this fungus.In summary,the proteogenomic strategy which we employed in this study allowed us to provide the high-quality comprehensive annotation of the A.flavus genome.We next conducted the experimental validation of the novel phosphoinositide kinase in order to explore its functional relevance in this microbial species.Our functional studies revealed that deletion of Fab1 could influence the growth,development,aflatoxin production,pathogenicity and vacuole/vesicle function in A.flavus.This study reveals novel insights into the molecular mechanisms whereby this pathogen produces aflatoxin and provides an important theoretical basis for the earlier prevention and control of aflatoxin hazards.This also enables us to understand the important role of Fab1 in other filamentous fungi. |
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