FgHtf1 Regulates Global Gene Expression towards Aerial Mycelium and Conidiophore Formation in the Cereal Fungal Pathogen Fusarium graminearum

Abstract

The homeobox gene family of transcription factors (HTF) controls many developmental pathways and physiological processes in eukaryotes. We previously showed that a conserved HTF in the plant-pathogenic fungus Fusarium graminearum, Htf1 (FgHtf1), regulates conidium morphology in that organism. This study investigated the mechanism of FgHtf1-mediated regulation and identified putative FgHtf1 target genes by a chromatin immunoprecipitation assay combined with parallel DNA sequencing (ChIP-seq) and RNA sequencing. A total of 186 potential binding peaks, including 142 genes directly regulated by FgHtf1, were identified. Subsequent motif prediction analysis identified two DNA-binding motifs, TAAT and CTTGT. Among the FgHtf1 target genes were FgHTF1 itself and several important conidiation-related genes (e.g., FgCON7), the chitin synthase pathway genes, and the aurofusarin biosynthetic pathway genes. In addition, FgHtf1 may regulate the cAMP-protein kinase A (PKA)-Msn2/4 and Ca²⁺-calcineurin-Crz1 pathways. Taken together, these results suggest that, in addition to autoregulation, FgHtf1 also controls global gene expression and promotes a shift to aerial growth and conidiation in F. graminearum by activation of FgCON7 or other conidiation-related genes.IMPORTANCE The homeobox gene family of transcription factors is known to be involved in the development and conidiation of filamentous fungi. However, the regulatory mechanisms and downstream targets of homeobox genes remain unclear. FgHtf1 is a homeobox transcription factor that is required for phialide development and conidiogenesis in the plant pathogen F. graminearum In this study, we identified FgHtf1-controlled target genes and binding motifs. We found that, besides autoregulation, FgHtf1 also controls global gene expression and promotes conidiation in F. graminearum by activation of genes necessary for aerial growth, FgCON7, and other conidiation-related genes.

Keywords: ChIP-seq; FgHtf1; Fusarium graminearum; RNA-seq; conidiogenesis.

FIG 1

FgHTF1 is a transcriptional activator. The BD constructs were transformed with pGADT7 (section 3, pGADT7 and pGBKT7-FgHtf1) into yeast strain Y187, which was also transformed with the positive control (section 1, pGADT7-T and pGBKT7-p53) and the negative control (section 2, pGADT7-T and pGBKT7-lam). Three independent experiments were performed, and each yielded a similar result.

FIG 2

Differentially expressed genes in the ΔFghtf1 mutant. Distribution of expression levels for annotated transcripts of the wild-type strain PH-1 and the ΔFghtf1 mutant. The gene distributions and expression level (log₂ ratio ≥ 1) are also shown. Significantly differentially expressed transcripts recorded between PH-1 and the ΔFghtf1 mutant are plotted in different colors (red, increased gene expression; blue, nondifferentially expressed genes; green, decreased gene expression). Pearson’s correlation coefficient (R²) between replicates was 0.897. TPM, transcripts per million clean tags.

FIG 3

Establishment of chromatin immunoprecipitation. The sequential work flow of ChIP-seq to identify FgHtf1 target genes in F. graminearum is shown. The complemented strain harboring GFP was used as the test sample. The mutant strain was used as the negative control. Sonicated DNA fragments that were not immunoprecipitated were used as the input control to test the quality of the sonicated DNA.

FIG 4

ChIP-qPCR was performed to validate the ChIP-seq results for select target genes. (A) ChIP-qPCR was performed to validate the ChIP-seq results. FGSG_10565, FGSG_02320, and FGSG_00800 were selected as specific targets from the ChIP-seq results, while three other genes (FGSG_09530, FGSG_07629, and FGSG_10523), including the β-tubulin gene, were analyzed as nonspecific targets. Data are presented as the means and standard errors from three biological replicates. (B) ChIP-qPCR was performed to validate the ChIP-seq results for three transcription factors, FGSG_07097 (FgHtf1), FGSG_10129 (FgStuA), and FGSG_04134 (FgCon7). Data are presented as the means and standard errors from three biological replicates. The β-tubulin gene (FGSG_09530) promoter was used as a nonspecific target.

FIG 5

Putative FgHtf1 binding motifs. The top 2 motifs (TAAT and CTTGT) were identified by use of the MEME Suite (version 4.11.1). The CentriMo algorithm was used to identify known or user-provided motifs. The DREME algorithm was used to discover the short, ungapped motifs that are relatively enriched in the ChIPed DNA sequence.

FIG 6

Proposed model of the pathway of FgHtf1 regulation. FgHtf1 regulates the transcription of itself and several genes for other pathways, including those for the conidiation-related, chitin synthase, aurofusarin biosynthetic, and cAMP-Ca²⁺-calcineurin-Crz1 pathways.