The FgHOG1 Pathway Regulates Hyphal Growth,Stress Responses,and Plant Infection in Fusarium graminearum

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.     

The Sch9 Kinase Regulates Conidium Size,Stress Responses,and Pathogenesis in Fusarium graminearum

Fusarium head blight caused by Fusarium graminearum is an important disease of wheat and barley worldwide. In a previous study on functional characterization of the F. graminearum kinome, one protein kinase gene important for virulence is orthologous to SCH9 that is functionally related to the cAMP-PKA and TOR pathways in the budding yeast. In this study, we further characterized the functions of FgSCH9 in F. graminearum and its ortholog in Magnaporthe oryzae. The ΔFgsch9 mutant was slightly reduced in growth rate but significantly reduced in conidiation, DON production, and virulence on wheat heads and corn silks. It had increased tolerance to elevated temperatures but became hypersensitive to oxidative, hyperosmotic, cell wall, and membrane stresses. The ΔFgsch9 deletion also had conidium morphology defects and produced smaller conidia. These results suggest that FgSCH9 is important for stress responses, DON production, conidiogenesis, and pathogenesis in F. graminearum. In the rice blast fungus Magnaporthe oryzae, the ΔMosch9 mutant also was defective in conidiogenesis and pathogenesis. Interestingly, it also produced smaller conidia and appressoria. Taken together, our data indicate that the SCH9 kinase gene may have a conserved role in regulating conidium size and plant infection in phytopathogenic ascomycetes.     

Network-Based Data Integration for Selecting Candidate Virulence Associated Proteins in the Cereal Infecting Fungus Fusarium graminearum

The identification of virulence genes in plant pathogenic fungi is important for understanding the infection process, host range and for developing control strategies. The analysis of already verified virulence genes in phytopathogenic fungi in the context of integrated functional networks can give clues about the underlying mechanisms and pathways directly or indirectly linked to fungal pathogenicity and can suggest new candidates for further experimental investigation, using a ‘guilt by association’ approach. Here we study 133 genes in the globally important Ascomycete fungus Fusarium graminearum that have been experimentally tested for their involvement in virulence. An integrated network that combines information from gene co-expression, predicted protein-protein interactions and sequence similarity was employed and, using 100 genes known to be required for virulence, we found a total of 215 new proteins potentially associated with virulence of which 29 are annotated as hypothetical proteins. The majority of these potential virulence genes are located in chromosomal regions known to have a low recombination frequency. We have also explored the taxonomic diversity of these candidates and found 25 sequences, which are likely to be fungal specific. We discuss the biological relevance of a few of the potentially novel virulence associated genes in detail. The analysis of already verified virulence genes in phytopathogenic fungi in the context of integrated functional networks can give clues about the underlying mechanisms and pathways directly or indirectly linked to fungal pathogenicity and can suggest new candidates for further experimental investigation, using a ‘guilt by association’ approach.     

The White Collar Complex Is Involved in Sexual Development of Fusarium graminearum

Sexual spores (ascospores) of Fusarium graminearum, a homothallic ascomycetous fungus, are believed to be the primary inocula for epidemics of the diseases caused by this species in cereal crops. Based on the light requirement for the formation of fruiting bodies (perithecia) of F. graminearum under laboratory conditions, we explored whether photoreceptors play an important role in sexual development. Here, we evaluated the roles of three genes encoding putative photoreceptors [a phytochrome gene (FgFph) and two white collar genes (FgWc-1 and FgWc-2)] during sexual development in F. graminearum. For functional analyses, we generated transgenic strains lacking one or two genes from the self-fertile Z3643 strain. Unlike the wild-type (WT) and add-back strains, the single deletion strains (ΔFgWc-1 and ΔFgWc-2) produced fertile perithecia under constant light on complete medium (CM, an unfavorable medium for sexual development) as well as on carrot agar (a perithecial induction condition). The expression of mating-type (MAT) genes increased significantly in the gene deletion strains compared to the WT under both conditions. Deletion of FgFph had no significant effect on sexual development or MAT gene expression. In contrast, all of the deletion strains examined did not show significant changes in other traits such as hyphal growth, mycotoxin production, and virulence. A split luciferase assay confirmed the in vivo protein-protein interactions among three photoreceptors along with FgLaeA, a global regulator of secondary metabolism and fungal development. Introduction of an intact copy of the A. nidulans LreA and LreB genes, which are homologs of FgWc-1 and FgWc-2, into the ΔFgWc-1 and ΔFgWc-2 strains, respectively, failed to repress perithecia formation on CM in the gene deletion strains. Taken together, these results demonstrate that FgWc-1 and FgWc-2, two central components of the blue-light sensing system, negatively regulate sexual development in F. graminearum, which differs from the regulation pattern in A. nidulans.     

Precocene II,a Trichothecene Production Inhibitor,Binds to Voltage-Dependent Anion Channel and Increases the Superoxide Level in Mitochondria of Fusarium graminearum

Precocene II, a constituent of essential oils, shows antijuvenile hormone activity in insects and inhibits trichothecene production in fungi. We investigated the molecular mechanism by which precocene II inhibits trichothecene production in Fusarium graminearum, the main causal agent of Fusarium head blight and trichothecene contamination in grains. Voltage-dependent anion channel (VDAC), a mitochondrial outer membrane protein, was identified as the precocene II-binding protein by an affinity magnetic bead method. Precocene II increased the superoxide level in mitochondria as well as the amount of oxidized mitochondrial proteins. Ascorbic acid, glutathione, and α-tocopherol promoted trichothecene production by the fungus. These antioxidants compensated for the inhibitory activity of precocene II on trichothecene production. These results suggest that the binding of precocene II to VDAC may cause high superoxide levels in mitochondria, which leads to stopping of trichothecene production.     

The Wor1-like Protein Fgp1 Regulates Pathogenicity,Toxin Synthesis and Reproduction in the Phytopathogenic Fungus Fusarium graminearum

WOR1 is a gene for a conserved fungal regulatory protein controlling the dimorphic switch and pathogenicity determents in Candida albicans and its ortholog in the plant pathogen Fusarium oxysporum, called SGE1, is required for pathogenicity and expression of key plant effector proteins. F. graminearum, an important pathogen of cereals, is not known to employ switching and no effector proteins from F. graminearum have been found to date that are required for infection. In this study, the potential role of the WOR1-like gene in pathogenesis was tested in this toxigenic fungus. Deletion of the WOR1 ortholog (called FGP1) in F. graminearum results in greatly reduced pathogenicity and loss of trichothecene toxin accumulation in infected wheat plants and in vitro. The loss of toxin accumulation alone may be sufficient to explain the loss of pathogenicity to wheat. Under toxin-inducing conditions, expression of genes for trichothecene biosynthesis and many other genes are not detected or detected at lower levels in Δfgp1 strains. FGP1 is also involved in the developmental processes of conidium formation and sexual reproduction and modulates a morphological change that accompanies mycotoxin production in vitro. The Wor1-like proteins in Fusarium species have highly conserved N-terminal regions and remarkably divergent C-termini. Interchanging the N- and C- terminal portions of proteins from F. oxysporum and F. graminearum resulted in partial to complete loss of function. Wor1-like proteins are conserved but have evolved to regulate pathogenicity in a range of fungi, likely by adaptations to the C-terminal portion of the protein.     

Roles of Cch1 and Mid1 in Morphogenesis, Oxidative Stress Response and Virulence in Candida albicans

Ca²⁺ channel Cch1, and its subunit Mid1, has been suggested as the protein complex responsible for mediating Ca²⁺ influx, which is often employed by fungal cells to maintain cell survival. The abilities of morphological switch and response to stress conditions are closely related to pathogenicity in Candida albicans. Cch1 and Mid1 activity are required for virulence of Cryptococcus neoformans and Claviceps purpurea, respectively. To investigate whether Cch1 and Mid1 also play a role in the virulence of C. albicans, we constructed cch1Δ/Δ and mid1Δ/Δ mutant strains for functional analysis of CCH1 and MID1. Although both of the mutants displayed the ability of yeast-to-hypha transition, they were defective in hyphae maintenance and invasive growth. Interestingly, deletion of CCH1 or MID1 in C. albicans led to an obvious defect phenotype in oxidative stress response. Moreover, the virulence of the mutants was reduced in a mouse model. Our results demonstrated that Cch1 and Mid1 activity are related to the virulence of C. albicans and may provide a new antifungal target.