Figure 1.
Phylogenetic analysis with the conventional protein kinase (ePK) genes of Fusarium graminearum.
The unrooted maximum likelihood tree was constructed with the catalytic domain sequences. Different protein kinase groups, AGC (PKA, PKG, PKC, βARK, ribosomal S6 family PKs and their close relatives), CAMK (calmodulin-regulated kinases), CK1 (casein kinase 1 and close relatives), CMGC (CDKs, MAPKs, GSK, and CDK-like kinases), STE (many kinases involved in the MAPK cascades), and Other (with a conserved kinase domain but could not be classified) are labeled with different colors. Fg05135 contains two catalytic domains (a and b).
Table 1.
Protein kinase genes essential in S. cerevisiae, S. pombe, or F. graminearum.
Figure 2.
Representative PK mutants with defects in colony morphology and hyphal growth.
A. Three-day-old PDA cultures of PH-1 and the Fg10037, Fg06957, Fg10066, Fg10381, Fg08635, Fg07344, and Fg08468. B. Hyphae of PH-1 and the Fg10066, Fg06957, Fg04947, Fg10037, and Fg10381 mutants grown on CM slab agars for 36 h. Bar = 100 µm.
Table 2.
Protein kinase genes important for vegetative growth and/or pathogenesis.
Figure 3.
Mutants with defects in conidiation and conidium morphology.
A. Five-day-old CMC cultures of PH-1 and the Fg01312, Fg08731, Fg08635, Fg07344, and Fg10381 mutants with defects in conidiogenesis. Arrows point to phialides and conidiophores. B. Conidia of PH-1 and the Fg04382, Fg06939, Fg04053, Fg10228, Fg06957, and Fg07329 mutants with defects in conidium morphology. Arrows point to foot cells. C. Conidia of PH-1 and the Fg06957, Fg07329, and Fg10381 mutants were stained with DAPI and Calcofluor. Bar = 20 µm.
Table 3.
Phenotypes of the 96 protein kinase mutants in sexual reproduction.
Figure 4.
Protein kinase mutants with defects in sexual reproduction.
All mating cultures were incubated at 25°C under black light. A. Asci and ascospores were not formed in perithecia produced by the Fg09612, Fg06793, Fg08468, Fg07344, and Fg10228 mutants. B. Ascospores of the Fg09274, Fg07251, Fg01058, and Fg01641 mutants. Bar = 20 µm.
Figure 5.
Mutants with altered sensitivity to hyperosmotic stress.
A. Colonies of PH-1 and the Fg00408, Fg08691, Fg09612, Fg06939, Fg04382, Fg09274m Fg09897, Fg04947, Fg01641, Fg04484, Fg08906 mutants formed on CM with (upper row) or without (lower row) 0.7 M NaCl after incubation for 3 days. B. Defects in hyphal growth of the Fg06939, Fg08691, and Fg00408 mutants in the presence of 0.7 M NaCl. C. Cultures of PH-1, Fg09612, Fg06939, Fg09274, Fg01641, and Fg08906 grown on CM with 0.7 M KCl or 1 M sorbitol.
Figure 6.
Mutants with defects in response to oxidative stress.
Colonies formed by the wild type (PH-1) and Fg04382, Fg13318, Fg05734, and Fg08701 mutants on PDA with (upper) or without (lower) 0.05% H2O2 after incubation for 5 days.
Figure 7.
Infection assays with flowering wheat heads.
A. Typical wheat heads infected with the wild-type strain PH-1 and mutants blocked in the three MAPK pathways. B. Categories of mutants with different disease indices.
Figure 8.
The predicted interactome of protein kinases in F. graminearum.
Orthologs of yeast protein kinases (PKs) were identified and used for the prediction of protein-protein interactions. The PK-PK interaction map was generated with Cytoscape. Three MAP kinase pathways are highlighted in green.
Figure 9.
Verification of predicted PK-PK interactions.
A. Different concentrations of yeast cells (cells/ml) of the transformants expressing the labeled bait and prey constructs were assayed for growth on SD-Leu-Trp-His plates. P and N were the positive and negative controls provided in the BD Matchmaker library construct kit. B. The same set of yeast transformants were assayed for β-galactosidase activities. C. Co-IP assays. Western blots of total proteins isolated from transformants expressing the GFP and 3xFLAG fusion constructs as labeled and proteins eluted from anti-FLAG beads were detected with a monoclonal anti-GFP antibody.
Figure 10.
Septation defects in the Fg10228, Fg11812, and Fg10381 mutants.
Germ tubes of the wild type and mutant strains were incubated at 25°C for 18 h. The same fields were observed under DIC (left) and epifluoresence (UV) microscopy. Bar = 20 µm. Arrows point to septa. Septum formation was irregular in the Fg11812 mutant, but the Fg10381 and Fg10228 mutants rarely produced septa.