The Transcription Factor FgAtrR Regulates Asexual and Sexual Development, Virulence, and DON Production and Contributes to Intrinsic Resistance to Azole Fungicides in Fusarium graminearum

Abstract

Fusarium graminearum is the predominant causal agent of cereal Fusarium head blight disease (FHB) worldwide. The application of chemical fungicides such as azole antifungals is still the primary method for FHB control. However, to date, our knowledge of transcriptional regulation in the azole resistance of F. graminearum is quite limited. In this study, we identified and functionally characterized a Zn(II)2-Cys6 transcription factor FgAtrR in F. graminearum. We constructed a FgAtrR deletion mutant and found that deletion of FgAtrR resulted in faster radial growth with serious pigmentation defects, significantly reduced conidial production, and an inability to form perithecia. The pathogenicity of the ΔFgAtrR mutant on wheat spikes and corn silks was severely impaired with reduced deoxynivalenol production, while the tolerance to prochloraz and propiconazole of the deletion mutant was also significantly decreased. RNA-seq indicated that many metabolic pathways were affected by the deletion of FgAtrR. Importantly, FgAtrR could regulate the expression of the FgCYP51A and ABC transporters, which are the main contributors to azole resistance. These results demonstrated that FgAtrR played essential roles in asexual and sexual development, DON production, and pathogenicity, and contributed to intrinsic resistance to azole fungicides in F. graminearum. This study will help us improve the understanding of the azole resistance mechanism in F. graminearum.

Keywords: Fusarium head blight; fungicide resistance; mycotoxin; plant pathogen; transcriptional regulation.

Figure 1

Domain structures and phylogenetic analysis. (a) Protein domains of FgAtrR and AfAtrR found in the Pfam database are shown. (b) The phylogenetic tree based on the amino acid sequence was constructed by the MEGA-X program using the neighboring-join method. The amino acid sequences from different species (shown in brackets) were aligned by MUSCLE.

Figure 2

FgAtrR is involved in vegetative growth, pigmentation, and asexual and sexual reproduction in F. graminearum. (a) Colony morphology of the wild-type strain PH-1, the ΔFgAtrR mutants, and ComFgAtrR strain. Strains were grown on PDA plates at 25 °C for 4 days. (b) The radial growth rate on PDA plates of the wild-type strain PH-1, ΔFgAtrR, and ComFgAtrR. These strains were cultured on PDA plates for 4 days at 25 °C, and the colony diameters of each strain were measured every 24 h. (c) The radial growth rate on SNA plates of the wild-type strain PH-1, ΔFgAtrR, and ComFgAtrR. These strains were cultured on PDA plates for 6 days at 25 °C, and the colony diameters of each strain were measured every 24 h. (d) Relative expression levels of pigment biosynthesis-related genes PKS12, AurJ, and AurF in each strain. The total RNA of each strain was extracted from mycelia collected after 48 h of incubation at 25 °C in PDB broth. The actin gene was used as the internal control, and the expression level of each gene in PH-1 was arbitrarily set to 1. (e) Perithecia and ascospore formation. Strains were first cultured on carrot agar for 7 d and then mock-fertilized to induce sexual reproduction using Tween 60 solution. Perithecia formation was examined after incubation for an additional 3 weeks. (f) Conidia production of each strain. Conidia were harvested from the CMC cultures after incubation at 25 °C and 180 rpm for 4 days. All experiments were repeated three times with three replicates each time. Linear bars denote standard errors of three experiments. The different letters on the bars indicate a significant difference at the p < 0.05 level.

Figure 3

FgAtrR is required for full virulence of Fusarium graminearum. (a) The representative infected wheat heads were examined at 14 dpi with a 2 mm diameter mycelial plug of each strain. (b) The disease index was determined by the number of symptomatic spikelets per wheat head at 14 dpi. Ten flowering wheat heads were inoculated for each strain. Error bars showed the standard deviation. The same letters indicate no statistically significant difference at the p < 0.05 level.

Figure 4

FgAtrR is vital for deoxynivalenol (DON) biosynthesis in Fusarium graminearum. (a) DON production level (per gram of dried mycelia) of each strain in TBI medium was measured. Error bars denote standard errors of three independent experiments. (b) Relative expression levels of DON biosynthesis-related genes TRI5, TRI6, and TRI11 in each strain. The actin gene was used as the internal control, and the expression level of each gene in PH-1 was arbitrarily set to 1. The means of bars followed by different letters are significantly different at the p < 0.05 level.

Figure 5

FgAtrR plays an important role in the response to azole fungicides of Fusarium graminearum. The EC50 value of prochloraz (a) and propiconazole (b) for F. graminearum PH-1, ΔFgAtrR, and ComFgAtrR strains were measured. Relative expression levels of azole fungicide target genes CYP51A, CYP51B, and CYP51C in each strain were also determined (c). The actin gene was used as the internal control, and the expression level of each gene in PH-1 was arbitrarily set to 1.

Figure 6

Subcellular localization of FgAtrR-GFP in Fusarium graminearum. Nuclei were stained with DAPI. The GFP and DAPI signals were examined under a fluorescence microscope. BF, bright field.

Figure 7

Transcriptome analysis of Fusarium graminearum wild-type strain PH-1 (WT) and FgAtrR deletion mutant strain (MT). (a) Volcano plot of identified transcripts. The dots in the upper left corner (labeled in green) are transcripts that were downregulated in the ΔFgAtrR strain, while the dots in the upper right corner (labeled in red) indicate those that were upregulated. (b) GO enrichment analysis of the downregulated genes in the ΔFgAtrR strain showed that 8 GO terms in molecular function (MF) were significantly enriched (p_adj < 0.05). Detailed data are listed in Table S3. (c) KEGG enrichment analysis of the downregulated genes in the ΔFgAtrR strain showed that 7 KEGG pathways were significantly enriched (p_adj < 0.05). Detailed data re listed in Table S4.