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Characterization of the CCAAT-binding transcription factor complex in the plant pathogenic fungus Fusarium graminearum.

ABSTRACT: The CCAAT sequence is a ubiquitous cis-element of eukaryotic promoters, and genes containing CCAAT sequences have been shown to be activated by the CCAAT-binding transcription factor complex in several eukaryotic model organisms. In general, CCAAT-binding transcription factors form heterodimers or heterotrimeric complexes that bind to CCAAT sequences within the promoters of target genes and regulate various cellular processes. To date, except Hap complex, CCAAT-binding complex has been rarely reported in fungi. In this study, we characterized two CCAAT-binding transcription factors (Fct1 and Fct2) in the plant pathogenic fungus Fusarium graminearum. Previously, FCT1 and FCT2 were shown to be related to DNA damage response among eight CCAAT-binding transcription factors in F. graminearum. We demonstrate that the nuclear CCAAT-binding complex of F. graminearum has important functions in various fungal developmental processes, not just DNA damage response but virulence and mycotoxin production. Moreover, the results of biochemical and genetic analyses revealed that Fct1 and Fct2 may form a complex and play distinct roles among the eight CCAAT-binding transcription factors encoded by F. graminearum. To the best of our knowledge, the results of this study represent a substantial advancement in our understanding of the molecular mechanisms underlying the functions of CCAAT-binding factors in eukaryotes.

SUBMITTER: Kim JE

PROVIDER: S-EPMC7078317 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Characterization of the CCAAT-binding transcription factor complex in the plant pathogenic fungus Fusarium graminearum.

Kim Jung-Eun JE Nam Hyejin H Park Jiyeun J Choi Gyung Ja GJ Lee Yin-Won YW Son Hokyoung H

Scientific reports 20200317 1

The CCAAT sequence is a ubiquitous cis-element of eukaryotic promoters, and genes containing CCAAT sequences have been shown to be activated by the CCAAT-binding transcription factor complex in several eukaryotic model organisms. In general, CCAAT-binding transcription factors form heterodimers or heterotrimeric complexes that bind to CCAAT sequences within the promoters of target genes and regulate various cellular processes. To date, except Hap complex, CCAAT-binding complex has been rarely re ...[more]

PMID: 32184445

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Project description:Lipases, which catalyze the hydrolysis of long-chain triglycerides, diglycerides, and monoglycerides into free fatty acids and glycerol, participate in various biological pathways in fungi. In this study, we examined the biological functions and regulatory mechanisms of fungal lipases via two approaches. First, we performed a systemic functional characterization of 86 putative lipase-encoding genes in the plant-pathogenic fungus Fusarium graminearum. The phenotypes were assayed for vegetative growth, asexual and sexual reproduction, stress responses, pathogenicity, mycotoxin production, and lipase activity. Most mutants were normal in the assessed phenotypes, implying overlapping roles for lipases in F. graminearum. In particular, FgLip1 and Fgl1 were revealed as core extracellular lipases in F. graminearum. Second, we examined the lipase activity of previously constructed transcription factor (TF) mutants of F. graminearum and identified three TFs and one histone acetyltransferase that significantly affect lipase activity. The relative transcript levels of FgLIP1 and FGL1 were markedly reduced or enhanced in these TF mutants. Among them, Gzzc258 was identified as a key lipase regulator that is also involved in the induction of lipase activity during sexual reproduction. To our knowledge, this study is the first comprehensive functional analysis of fungal lipases and provides significant insights into the genetic and regulatory mechanisms underlying lipases in fungi. IMPORTANCE Fusarium graminearum is an economically important plant-pathogenic fungus that causes Fusarium head blight (FHB) on wheat and barley. Here, we constructed a gene knockout mutant library of 86 putative lipase-encoding genes and established a comprehensive phenotypic database of the mutants. Among them, we found that FgLip1 and Fgl1 act as core extracellular lipases in this pathogen. Moreover, several putative transcription factors (TFs) that regulate the lipase activities in F. graminearum were identified. The disruption mutants of F. graminearum-lipase regulatory TFs all showed defects in sexual reproduction, which implies a strong relationship between sexual development and lipase activity in this fungus. These findings provide valuable insights into the genetic mechanisms regulating lipase activity as well as its importance to the developmental stages of this plant-pathogenic fungus.

Project description:Fusarium graminearum is an important plant-pathogenic fungus that causes Fusarium head blight on wheat and barley, and ear rot on maize worldwide. This fungus has been widely used as a model organism to study various biological processes of plant-pathogenic fungi because of its amenability to genetic manipulation and well-established outcross system. Gene deletion and overexpression/constitutive expression of target genes are tools widely used to investigate the molecular mechanism underlying fungal development, virulence, and secondary metabolite production. However, for fine-tuning gene expression and studying essential genes, a conditional gene expression system is necessary that enables repression or induction of gene expression by modifying external conditions. Until now, only a few conditional expression systems have been developed in plant-pathogenic fungi. This study proposes a new and versatile conditional gene expression system in F. graminearum using the promoter of a copper-responsive gene, designated F. graminearum copper-responsive 1 (FCR1). Transcript levels of FCR1 were found to be greatly affected by copper availability conditions. Moreover, the promoter (PFCR1 ), 1 kb upstream of the FCR1 open reading frame, was sufficient to confer copper-dependent gene expression. Replacement of a green fluorescent protein gene and FgENA5 promoter with a PFCR1 promoter clearly showed that PFCR1 could be used for fine-tuning gene expression in this fungus. We also demonstrated the applicability of this conditional gene expression system to an essential gene study by replacing the promoter of FgIRE1, an essential gene of F. graminearum. This enabled the generation of FgIRE1 suppression mutants, which allowed functional characterization of the gene. This study reported the first conditional gene expression system in F. graminearum using both repression and induction. This system would be a convenient way to precisely control gene expression and will be used to determine the biological functions of various genes, including essential ones.

Project description:BackgroundAccurate genome assembly and gene model annotation are critical for comparative species and gene functional analyses. Here we present the completed genome sequence and annotation of the reference strain PH-1 of Fusarium graminearum, the causal agent of head scab disease of small grain cereals which threatens global food security. Completion was achieved by combining (a) the BROAD Sanger sequenced draft, with (b) the gene predictions from Munich Information Services for Protein Sequences (MIPS) v3.2, with (c) de novo whole-genome shotgun re-sequencing, (d) re-annotation of the gene models using RNA-seq evidence and Fgenesh, Snap, GeneMark and Augustus prediction algorithms, followed by (e) manual curation.ResultsWe have comprehensively completed the genomic 36,563,796 bp sequence by replacing unknown bases, placing supercontigs within their correct loci, correcting assembly errors, and inserting new sequences which include for the first time complete AT rich sequences such as centromere sequences, subtelomeric regions and the telomeres. Each of the four F. graminearium chromosomes was found to be submetacentric with respect to centromere positioning. The position of a potential neocentromere was also defined. A preferentially higher frequency of genetic recombination was observed at the end of the longer arm of each chromosome. Within the genome 1529 gene models have been modified and 412 new gene models predicted, with a total gene call of 14,164. The re-annotation impacts upon 69 entries held within the Pathogen-Host Interactions database (PHI-base) which stores information on genes for which mutant phenotypes in pathogen-host interactions have been experimentally tested, of which 59 are putative transcription factors, 8 kinases, 1 ATP citrate lyase (ACL1), and 1 syntaxin-like SNARE gene (GzSYN1). Although the completed F. graminearum contains very few transposon sequences, a previously unrecognised and potentially active gypsy-type long-terminal-repeat (LTR) retrotransposon was identified. In addition, each of the sub-telomeres and centromeres contained either a LTR or MarCry-1_FO element. The full content of the proposed ancient chromosome fusion sites has also been revealed and investigated. Regions with high recombination previously noted to be rich in secretome encoding genes were also found to be rich in tRNA sequences. This study has identified 741 F. graminearum species specific genes and provides the first complete genome assembly for a Sordariomycetes species.ConclusionsThis fully completed F. graminearum PH-1 genome and manually curated annotation, available at Ensembl Fungi, provides the optimum resource to perform interspecies comparative analyses and gene function studies.

Project description:Fusarium graminearum (teleomorph: Ascomycota, Hypocreales, Gibberella, Gibberella zeae) is a destructive fungal pathogen that threatens the production and quality of wheat and barley worldwide. Controlling this toxin-producing pathogen is a significant challenge. In the present study, the commercially available strain Bacillus amyloliquefaciens (Bacteria, Firmicutes, Bacillales, Bacillus) FZB42 showed strong activity against F. graminearum The lipopeptide bacillomycin D, produced by FZB42, was shown to contribute to the antifungal activity. Purified bacillomycin D showed strong activity against F. graminearum, and its 50% effective concentration was determined to be approximately 30 μg/ml. Analyses using scanning and transmission electron microscopy revealed that bacillomycin D caused morphological changes in the plasma membranes and cell walls of F. graminearum hyphae and conidia. Fluorescence microscopy combined with different dyes showed that bacillomycin D induced the accumulation of reactive oxygen species and caused cell death in F. graminearum hyphae and conidia. F. graminearum secondary metabolism also responded to bacillomycin D challenge, by increasing the production of deoxynivalenol. Biological control experiments demonstrated that bacillomycin D exerted good control of F. graminearum on corn silks, wheat seedlings, and wheat heads. In response to bacillomycin D, F. graminearum genes involved in scavenging reactive oxygen species were downregulated, whereas genes involved in the synthesis of deoxynivalenol were upregulated. Phosphorylation of MGV1 and HOG1, the mitogen-activated protein kinases of F. graminearum, was increased in response to bacillomycin D. Taken together, these findings reveal the mechanism of the antifungal action of bacillomycin D.IMPORTANCE Biological control of plant disease caused by Fusarium graminearum is desirable. Bacillus amyloliquefaciens FZB42 is a representative of the biocontrol bacterial strains. In this work, the lipopeptide bacillomycin D, produced by FZB42, showed strong fungicidal activity against F. graminearum Bacillomycin D caused morphological changes in the plasma membrane and cell wall of F. graminearum, induced accumulation of reactive oxygen species, and ultimately caused cell death in F. graminearum Interestingly, when F. graminearum was challenged with bacillomycin D, the deoxynivalenol production, gene expression, mitogen-activated protein kinase phosphorylation, and pathogenicity of F. graminearum were significantly altered. These findings clarified the mechanisms of the activity of bacillomycin D against F. graminearum and highlighted the potential of B. amyloliquefaciens FZB42 as a biocontrol agent against F. graminearum.

Dataset Information

Characterization of the CCAAT-binding transcription factor complex in the plant pathogenic fungus Fusarium graminearum.

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Characterization of the CCAAT-binding transcription factor complex in the plant pathogenic fungus Fusarium graminearum.

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