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.     

Death-associated Protein 3 Regulates Mitochondrial-encoded Protein Synthesis and Mitochondrial Dynamics

Mitochondrial morphologies change over time and are tightly regulated by dynamic machinery proteins such as dynamin-related protein 1 (Drp1), mitofusion 1/2, and optic atrophy 1 (OPA1). However, the detailed mechanisms of how these molecules cooperate to mediate fission and fusion remain elusive. DAP3 is a mitochondrial ribosomal protein that involves in apoptosis, but its biological function has not been well characterized. Here, we demonstrate that DAP3 specifically localizes in the mitochondrial matrix. Knockdown of DAP3 in mitochondria leads to defects in mitochondrial-encoded protein synthesis and abnormal mitochondrial dynamics. Moreover, depletion of DAP3 dramatically decreases the phosphorylation of Drp1 at Ser-637 on mitochondria, enhancing the retention time of Drp1 puncta on mitochondria during the fission process. Furthermore, autophagy is inhibited in the DAP3-depleted cells, which sensitizes cells to different types of death stimuli. Together, our results suggest that DAP3 plays important roles in mitochondrial function and dynamics, providing new insights into the mechanism of a mitochondrial ribosomal protein function in cell death.     

Association between Virulence and Triazole Tolerance in the Phytopathogenic Fungus Mycosphaerella graminicola

Host resistance and synthetic antimicrobials such as fungicides are two of the main approaches used to control plant diseases in conventional agriculture. Although pathogens often evolve to overcome host resistance and antimicrobials, the majority of reports have involved qualitative host – pathogen interactions or antimicrobials targeting a single pathogen protein or metabolic pathway. Studies that consider jointly the evolution of virulence, defined as the degree of damage caused to a host by parasite infection, and antimicrobial resistance are rare. Here we compared virulence and fungicide tolerance in the fungal pathogen Mycosphaerella graminicola sampled from wheat fields across three continents and found a positive correlation between virulence and tolerance to a triazole fungicide. We also found that quantitative host resistance selected for higher pathogen virulence. The possible mechanisms responsible for these observations and their consequences for sustainable disease management are discussed.     

The Secreted Antifungal Protein Thionin 2.4 in Arabidopsis thaliana Suppresses the Toxicity of a Fungal Fruit Body Lectin from Fusarium graminearum

Plants possess active defense systems and can protect themselves from pathogenic invasion by secretion of a variety of small antimicrobial or antifungal proteins such as thionins. The antibacterial and antifungal properties of thionins are derived from their ability to induce open pore formation on cell membranes of phytopathogens, resulting in release of potassium and calcium ions from the cell. Wheat thionin also accumulates in the cell walls of Fusarium-inoculated plants, suggesting that it may have a role in blocking pathogen infection at the plant cell walls. Here we developed an anti-thionin 2.4 (Thi2.4) antibody and used it to show that Thi2.4 is localized in the cell walls of Arabidopsis and cell membranes of F. graminearum, when flowers are inoculated with F. graminearum. The Thi2.4 protein had an antifungal effect on F. graminearum. Next, we purified the Thi2.4 protein, conjugated it with glutathione-S-transferase (GST) and coupled the proteins to an NHS-activated column. Total protein from F. graminearum was applied to GST-Thi2.4 or Thi2.4-binding columns, and the fungal fruit body lectin (FFBL) of F. graminearum was identified as a Thi2.4-interacting protein. This interaction was confirmed by a yeast two-hybrid analysis. To investigate the biological function of FFBL, we infiltrated the lectin into Arabidopsis leaves and observed that it induced cell death in the leaves. Application of FFBL at the same time as inoculation with F. graminearum significantly enhanced the virulence of the pathogen. By contrast, FFBL-induced host cell death was effectively suppressed in transgenic plants that overexpressed Thi2.4. We found that a 15 kD Thi2.4 protein was specifically expressed in flowers and flower buds and suggest that it acts not only as an antifungal peptide, but also as a suppressor of the FFBL toxicity. Secreted thionin proteins are involved in this dual defense mechanism against pathogen invasion at the plant-pathogen interface.     

Circular DNA Plasmid in the Phytopathogenic Fungus Alternaria Alternata: Its Temperature-Dependent Curing and Association with Pathogenicity

We found the presence of plasmid DNA in strain T88-56 of the Japanese pear pathotype of Alternaria alternata, which causes black spot of certain cultivars of Japanese pear by producing host-specific AK-toxin. The plasmid, designated pAAT56, was identified to be an ~5.4-kilobase (kb) circular molecule by electron microscopic observation and restriction endonuclease mapping. Southern blot analysis showed that pAAT56 DNA had no homology with either nuclear or mitochondrial DNA. Cultures of strain T88-56 grown at 26° showed markedly reduced plasmid levels relative to those grown at lower temperatures. The strain was completely cured of pAAT56 during growth at 29°. Temperature-dependent curing of pAAT56 was confirmed by using single-protoplast isolates from mycelia grown at 23°, most of which maintained the plasmid, and from mycelia grown at 29°, most of which had lost the plasmid. Northern blot analysis detected the presence of three RNA species (~1.7, 2.7 and 5.4 kb) transcribed from pAAT56. The biological function of pAAT56 was observed using single-protoplast isolates from mycelia that either contained or had been cured of pAAT56. The plasmid-containing isolates tended to be reduced in AK-toxin production and pathogenicity compared with the plasmid-cured isolates.     

Two Cdc2 Kinase Genes with Distinct Functions in Vegetative and Infectious Hyphae in Fusarium graminearum

Eukaryotic cell cycle involves a number of protein kinases important for the onset and progression through mitosis, most of which are well characterized in the budding and fission yeasts and conserved in other fungi. However, unlike the model yeast and filamentous fungi that have a single Cdc2 essential for cell cycle progression, the wheat scab fungus Fusarium graminearum contains two CDC2 orthologs. The cdc2A and cdc2B mutants had no obvious defects in growth rate and conidiation but deletion of both of them is lethal, indicating that these two CDC2 orthologs have redundant functions during vegetative growth and asexual reproduction. However, whereas the cdc2B mutant was normal, the cdc2A mutant was significantly reduced in virulence and rarely produced ascospores. Although deletion of CDC2A had no obvious effect on the formation of penetration branches or hyphopodia, the cdc2A mutant was limited in the differentiation and growth of infectious growth in wheat tissues. Therefore, CDC2A plays stage-specific roles in cell cycle regulation during infectious growth and sexual reproduction. Both CDC2A and CDC2B are constitutively expressed but only CDC2A was up-regulated during plant infection and ascosporogenesis. Localization of Cdc2A- GFP to the nucleus but not Cdc2B-GFP was observed in vegetative hyphae, ascospores, and infectious hyphae. Complementation assays with chimeric fusion constructs showed that both the N- and C-terminal regions of Cdc2A are important for its functions in pathogenesis and ascosporogenesis but only the N-terminal region is important for its subcellular localization. Among the Sordariomycetes, only three Fusarium species closely related to F. graminearum have two CDC2 genes. Furthermore, F. graminearum uniquely has two Aurora kinase genes and one additional putative cyclin gene, and its orthologs of CAK1 and other four essential mitotic kinases in the budding yeast are dispensable for viability. Overall, our data indicate that cell cycle regulation is different between vegetative and infectious hyphae in F. graminearum and Cdc2A, possibly by interacting with a stage-specific cyclin, plays a more important role than Cdc2B during ascosporogenesis and plant infection.     

Deleted in Breast Cancer 1 (DBC1) Protein Regulates Hepatic Gluconeogenesis

Liver gluconeogenesis is essential to provide energy to glycolytic tissues during fasting periods. However, aberrant up-regulation of this metabolic pathway contributes to the progression of glucose intolerance in individuals with diabetes. Phosphoenolpyruvate carboxykinase (PEPCK) expression plays a critical role in the modulation of gluconeogenesis. Several pathways contribute to the regulation of PEPCK, including the nuclear receptor Rev-erbα and the histone deacetylase SIRT1. Deleted in breast cancer 1 (DBC1) is a nuclear protein that binds to and regulates both Rev-erbα and SIRT1 and, therefore, is a candidate to participate in the regulation of PEPCK. In this work, we provide evidence that DBC1 regulates glucose metabolism and the expression of PEPCK. We show that DBC1 levels decrease early in the fasting state. Also, DBC1 KO mice display higher gluconeogenesis in a normal and a high-fat diet. DBC1 absence leads to an increase in PEPCK mRNA and protein expression. Conversely, overexpression of DBC1 results in a decrease in PEPCK mRNA and protein levels. DBC1 regulates the levels of Rev-erbα, and manipulation of Rev-erbα activity or levels prevents the effect of DBC1 on PEPCK. In addition, Rev-erbα levels decrease in the first hours of fasting. Finally, knockdown of the deacetylase SIRT1 eliminates the effect of DBC1 knockdown on Rev-erbα levels and PEPCK expression, suggesting that the mechanism of PEPCK regulation is, at least in part, dependent on the activity of this enzyme. Our results point to DBC1 as a novel regulator of gluconeogenesis.