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.

Figure 1

Characterization of the nuclear CCAAT-binding complex of F. graminearum. (a) Nuclear localization of Fct1-GFP and Fct2-GFP. FCT1c-r/FCT2c-r strains carrying both Fct1-GFP or Fct2-GFP and hH1-RFP were used for the colocalization study. Scale bar = 20 µm. (b) Consensus binding sequence identified via the PBM assay. Consensus sequences that robustly bound to the Fct2-DsRed fusion protein. (c) The effects of positional mutations in CCAATC. To visualize the effects of the mutations on the binding intensities in the consensus binding motif, the average binding intensities (+) of the probes containing the core consensus 6-mer binding motif CCAATC relative to those of probes with mutations at each position (bar) are plotted. (d) Yeast two-hybrid analysis of the interaction between Fct1 and Fct2. The plasmid pairs pDHB1-Fct1/pAI-Alg5 and pDHB1-Fct1/pDL2-Alg5 served as positive and negative controls, respectively. The growth of the transformed yeast was assayed on synthetic dextrose medium lacking Leu and Trp (SD-LT) or Leu, Trp, His, and Ade (SD-LTHA). The columns in each panel represent serial decimal dilutions.

Figure 2

Phylogenetic tree of fungal CCAAT-binding factors. The alignment was performed with ClustalW, and MEGA X was used to perform a 1,000-bootstrap phylogenetic analysis using the neighbour joining method. Bootstrap support is shown for each node.

Figure 3

Vegetative growth and sexual development of F. graminearum strains. (a) The mycelial growth of F. graminearum strains on complete medium (CM), CM supplemented with 10 mM hydroxyurea (HU), 10 mU/ml bleomycin (BLM), and minimal medium (MM). The strains were imaged 5 days after inoculation. (b) Sexual development. A five-day-old culture on carrot agar medium was mock-fertilized to induce sexual reproduction, and the cultures were incubated for an additional 7 days. Scale bar = 500 µm.

Figure 4

Virulence of F. graminearum strains. (a) Virulence on wheat heads. The centre spikelet of each wheat head was injected with 10 μl of a conidial suspension. Images were captured 21 days after inoculation. (b) Disease index. The disease index was estimated as the number of diseased spikelets on each wheat head. Asterisks represent significant difference between the wild-type strain and each mutant (p < 0.001). (c) Micrographs of manually generated sections after the infection of wheat. Wheat spikelets were inoculated with conidial suspensions from strains expressing GFP in the cytoplasm (HK12, fct1-g, fct2-g, and fct1/2-g). Infected wheat heads were longitudinally dissected 6 days after inoculation and examined under a fluorescence microscope. GFP fluorescence indicates hyphal spreading from the inoculation points. Arrowheads mark the inoculated spikelets. Reflected, reflected light.

Figure 5

Trichothecene production by the F. graminearum strains. (a) Total trichothecene production. Each strain was grown in minimal medium containing 5 mM agmatine (MMA) for 7 days. Trichothecenes were analysed via gas chromatography-mass spectrometry (GC-MS) and quantified based on the biomass of each strain. (b) Transcript levels of TRI5 and TRI6 in the F. graminearum strains. The transcript levels were analysed via quantitative real-time PCR (qRT-PCR) 4 days after inoculation in MMA. Asterisks represent significant differences in the relative transcript levels of TRI5 and TRI6 between the wild-type strain and each mutant (p < 0.001).