The FgHOG1 pathway regulates hyphal growth, stress responses, and plant infection in Fusarium graminearum

Abstract

Fusarium head blight (FHB) caused by Fusarium graminearum is a destructive disease of wheat and barley worldwide. In a previous study of systematic characterization of protein kinase genes in F. graminearum, mutants of three putative components of the osmoregulation MAP kinase pathway were found to have distinct colony morphology and hyphal growth defects on PDA plates. Because the osmoregulation pathway is not known to regulate aerial hyphal growth and branching, in this study we further characterized the functions of the FgHog1 pathway in growth, pathogenesis, and development. The Fghog1, Fgpbs2, and Fgssk2 mutants were all reduced in growth rate, aerial hyphal growth, and hyphal branching angle. These mutants were not only hypersensitive to osmotic stress but also had increased sensitivity to oxidative, cytoplasm membrane, and cell wall stresses. The activation of FgHog1 was blocked in the Fgpbs2 and Fgssk2 mutants, indicating the sequential activation of FgSsk2-FgPbs2-FgHog1 cascade. Interestingly, the FgHog1 MAPK pathway mutants appeared to be sensitive to certain compounds present in PDA. They were female sterile but retained male fertility. We also used the metabolomics profiling approach to identify compatible solutes that were accumulated in the wild type but not in the Fghog1 deletion mutant. Overall, our results indicate that the FgSsk2-FgPbs2-FgHog1 MAPK cascade is important for regulating hyphal growth, branching, plant infection, and hyperosmotic and general stress responses in F. graminearum.

Figure 1. Growth defects of the Fghog1, Fgpbs2, and Fgssk2 mutants.

A. Colonies of the wild type (PH-1) and the Fghog1 (HG15), Fgpbs2 (PS15), and Fgssk2 (FK13) mutants grown on PDA and 5xYEG agar plates for 3 days. B. Colony surface hydrophobicity tests with the same set of mutants. Photos were taken 15 min. after placing droplets of 50 µl red ink on the surface of the wild-type and mutant colonies. C. Hyphal tip growth and branching patterns of PH-1 and the same set of mutants on PDA plates. The branching angles were reduced in the extension zone of mutant colonies. Bar = 150 µm.

Figure 2. Defects of the Fghog1, Fgpbs2, and Fgssk2 mutants in response to hyperosmotic stress.

A. Colonies of PH-1, the Fghog1 (HG15), Fgpbs2 (PS15), and Fgssk2 (FK13) mutants, and the Fghog1/FgHOG1 complemented transformant (HGC1) on CM plates with or without 1 M NaCl. B. Conidium germination of PH-1 and the Fghog1 mutant in CM with 0.7 M NaCl examined at 3 h, 12 h, and 18 h. Bar = 20 µm. C. Germlings of PH-1, HG15, PS15, FK13, and HGC1 incubated in CM+1 M KCl for 12 h. Bar = 40 µm.

Figure 3. The FgHog1 MAPK pathway also is involved in responses to oxidative, cytoplasm membrane, and cell wall stresses.

A. Colonies of PH-1 and the Fghog1 (HG15), Fgpbs2 (PS15), and Fgssk2 (FK13) mutants on media with 0.05% H₂O₂, SDS, and Congo red. B. Germ tubes of the Fghog1, Fgpbs2, and Fgssk2 mutants incubated in liquid YEPD with 0.005% H₂O₂. Bar = 40 µm.

Figure 4. Defects of the Fghog1, Fgpbs2, and Fgssk2 mutants in sexual reproduction.

A. Self-crossing cultures of the wild type (PH-1) and the Fghog1 (HG15), Fgpbs2 (PS15), and Fgssk2 (FK13) mutants. Fertile perithecia with cirrhi were only observed with the wild type. B. Carrot agar cultures of the mat2 mutant fertilized with Fghog1, Fgpbs2, and Fgssk2 mutants. All the mutants retained male fertility. The close-up views were taken under a dissecting microscope.

Figure 5. Flowering wheat heads were inoculated with the wild type (PH-1) and Fghog1 mutant (HG15).

A. Colonization of glume tissues by PH-1 and HG15 was examined 48 hpi. B. The rachises directly beneath the inoculated spikeletes were examined 120 hpi. Hyphae growth (marked with arrows) was abundant in plant tissues inoculated with PH-1 and but not in samples inoculated with Fghog1 mutant. Bar = 40 µm.

Figure 6. Corn stalks inoculated with the wild type (PH-1) and the Fghog1 (HG15), Fgpbs2 (PS15), and Fgssk2 (FK13) mutants were examined 10 dpi.

Arrows pointed to the inoculation sites.

Figure 7. Expression and subcellular localization of FgHog1-GFP.

A. Conidia harvested from the Fghog1/FgHOG1-GFP transformant HGC1 were re-suspended in sterile distilled water or 0.3 M NaCl and examined by DIC or epifluorescence microscopy (GFP). B. GFP signals in germlings of FGC1 were incubated in the liquid YEPD medium with or without 0.3M NaCl. Nuclei were stained with DAPI. Bar = 20 µm.

Figure 8. Assays for the activation of FgHog1, Mgv1, and Gpmk1 MAP kinases.

Total proteins were isolated from vegetative hyphae of the wild type (PH-1) and the Fghog1 (HG15), Fgpbs2 (PS15), and Fgssk2 (FK13) mutants. A. The anti-TpGY antibody was used to detect the phosphorylation of FgHog1 (41-kDa) in cultures treated with or without 0.7 M NaCl. B. Phosphorylation of Mgv1 (46-kDa) and Gpmk1 (42-kDa) was detected with the anti-TpEY antibody. Anti-actin and anti-MAPK antibodies were used to determine the same loading amount of protein.

Figure 9

Metabolic profiles of the wild type PH-1 (A), Fghog1 mutant (B), and the Fghog1/FgHOG1 complemented transformant (C) cultured in YEPD with or without 1 M NaCl. The X-axis is the retention time (RT) in minutes. The Y-axis represents the abundance of total ion current. The peaks with RT of 6.64, 20.43, 26.15, and 44.09 are glycerol, arabitol, mannitol, and sucrose, respectively. The far left peak (RT = 5.75) is 1,2,2,3,4,5-hexamethyl-1,2,5-azasilaborole.