Fusarium graminearum Ste3 G-Protein Coupled Receptor: A Mediator of Hyphal Chemotropism and Pathogenesis

Abstract

Fungal hyphal chemotropism has been shown to be a major contributor to host-pathogen interactions. Previous studies on Fusarium species have highlighted the involvement of the Ste2 G-protein-coupled receptor (GPCR) in mediating polarized hyphal growth toward host-released peroxidase. Here, the role of the opposite mating type GPCR, Ste3, is characterized with respect to Fusarium graminearum chemotropism and pathogenicity. Fgste3Δ deletion strains were found to be compromised in the chemotropic response toward peroxidase, development of lesions on germinating wheat, and infection of Arabidopsis thaliana leaves. In the absence of FgSte3 or FgSte2, F. graminearum cells exposed to peroxidase showed no phosphorylation of the cell-wall integrity, mitogen-activated protein kinase pathway component Mgv1. In addition, transcriptomic gene expression profiling yielded a list of genes involved in cellular reorganization, cell wall remodeling, and infection-mediated responses that were differentially modulated by peroxidase when FgSte3 was present. Deletion of FgSte3 yielded the downregulation of genes associated with mycotoxin biosynthesis and appressorium development, compared to the wild-type strain, both in the presence of peroxidase. Together, these findings contribute to our understanding of the mechanism underlying fungal chemotropism and pathogenesis while raising the novel hypothesis that FgSte2 and FgSte3 are interdependent on each other for the mediation of the redirection of hyphal growth in response to host-derived peroxidase. IMPORTANCE Fusarium head blight of wheat, caused by the filamentous fungus Fusarium graminearum, leads to devastating global food shortages and economic losses. Fungal hyphal chemotropism has been shown to be a major contributor to host-pathogen interactions. Here, the role of the opposite mating type GPCR, Ste3, is characterized with respect to F. graminearum chemotropism and pathogenicity. These findings contribute to our understanding of the mechanisms underlying fungal chemotropism and pathogenesis.

Keywords: Fusarium graminearum; Fusarium head blight; G-protein coupled receptors; hyphal chemotropism; pheromone receptor; wheat disease; wheat infection.

FIG 1

F. graminearum chemotropic growth toward peroxidase is mediated by both Ste3 and Ste2 receptors. Polarized hyphal growth of wild-type, Fgste3Δ-1, Fgste3Δ-3, Fgste3Δ-4, and Fgste2Δ-5 were calculated 12 h after exposure to the indicated chemoattractants. Hyphae growing toward either 4 μM horseradish peroxidase (HRP), pheromones a-factor or α-factor (378 μM) were counted against a competing solvent control gradient (water or 50% vol/vol methanol, respectively). Data are representative of averages of 3 independent replicates (n = 500 hyphae/interaction/replicate; **, P < 0.001). Error bars represent the standard deviation. The statistics were assessed using Student’s t test.

FIG 2

FgSTE3 deletion has no effect on F. graminearum morphology or on osmotic stress tolerance. (A) Images of wild-type and mutant strains Fgste2Δ-5, Fgste3Δ-1, Fgste3Δ-3, and Fgmgv1Δ conidia grown on PDA, Congo red (CR; 150 μg/mL), and NaCl (0.7 M). Similar results were obtained for two independent experiments. Size bar = 1 cm. (B) Conidia for the wild-type, Fgste3Δ-3 and Fgste3Δ-1 were imaged under 100× using oil immersion. The images were captured using cellSens software, version 1.12. Size bar = 10 μM.

FIG 3

Deletion of FgSTE3 leads to decreased F. graminearum pathogenicity against wheat. (A) The pathogenicity was quantified via the measurement of the length of infected stalk or lesion formed on germinating “Roblin” coleoptiles that were infected with the indicated F. graminearum strains. Shown are representations of lesions formed around the wound site 10 days after infection with F. graminearum conidia. (B) Quantification of average lesion length formed on germinating “Roblin” coleoptile stalks infected with F. graminearum wild-type and mutant strains Fgste3Δ-3 and Fgste2Δ-5. The averages of two representative experiments are shown (compared to the wild-type strain; n = 18; **, P < 0.005). Error bars represent the standard deviation. The statistics were assessed via Student’s t test. Size bar = 1 cm.

FIG 4

Deletion of FgSTE3 leads to decreased F. graminearum pathogenicity against A. thaliana. (A) Representative images of A. thaliana leaf infected with F. graminearum strains. (B) Assessment of lesion development on A. thaliana leaves. Leaves were infected with the wild-type, Fgste3Δ-3, and Fgste2Δ-5 strains and quantified 3 days postinfection using Image J software. (C) Assessment of F. graminearum infection via quantitative PCR. Quantitative PCR was performed with genomic DNA isolated from Arabidopsis leaves infected with the wild-type, Fgste3Δ-3, or Fgste2Δ-5 strain. The relative expression (RQ) was measured with the Fusarium ITS2, with respect to the Arabidopsis PP2A. The experiment was performed three times (three biological replicates with n = 12 for each) with similar results. Error bars denote the standard deviation. The statistical analysis was performed using Student’s t test (****, P < 0.0005). Size bar = 1 cm.

FIG 5

Activation of the CWI-MAPK pathway by peroxidase is mediated by FgSte3 and FgSte2. (A) Representative images of a quantitative Western blot to probe CWI pathway activation by tracking the phosphorylation of Mgv1 and total MAPK isolated from wild-type and mutant strains Fgste3Δ-3, Fgste2Δ-5, and Fgmgv1Δ. The conidia were grown for 48 h in regular PDB culture and were treated with commercially available HRP or a water control for 1 h before total protein extraction. For the normalization of the quantification, α-tubulin was probed. The molecular weights of the detected proteins are indicated on the blot. (B) The intensity of phospho-Mgv1 was quantified and normalized to tubulin, with relative intensities compared to the wild-type (***, P < 0.0005). Quantification analysis was performed using ImageJ software. The data represent averages of three independent experiments. Error bars represent the standard deviation. The statistical analysis performed using Student’s t test.

FIG 6

Transcriptomic overview of F. graminearum responses in the presence and absence of peroxidase and FgSte3. (A) Heat map showing the differential gene expression for different strains and conditions highlighting varying levels of upregulation and downregulation. (B) PCA plot showing the clustering of three biological replicates for each representative condition. (C) Volcano plot showing upregulated responses in the wild-type + HRP versus the wild-type uninduced pairwise comparison.