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Fusarium graminearum is a causal agent of Fusarium head blight (FHB) and a deoxynivalenol (DON) producer. In this study, OSP24 is identified as an important virulence factor in systematic characterization of the 50 orphan secreted protein (OSP) genes of F. graminearum. Although dispensable for growth and initial penetration, OSP24 is important for infectious growth in wheat rachis tissues. OSP24 is specifically expressed during pathogenesis and its transient expression suppresses BAX- or INF1-induced cell death. Osp24 is translocated into plant cells and two of its 8 cysteine-residues are required for its function. Wheat SNF1-related kinase TaSnRK1α is identified as an Osp24-interacting protein and shows to be important for FHB resistance in TaSnRK1α-overexpressing or silencing transgenic plants. Osp24 accelerates the degradation of TaSnRK1α by facilitating its association with the ubiquitin-26S proteasome. Interestingly, TaSnRK1α also interacts with TaFROG, an orphan wheat protein induced by DON. TaFROG competes against Osp24 for binding with the same region of TaSnRKα and protects it from degradation. Overexpression of TaFROG stabilizes TaSnRK1α and increases FHB resistance. Taken together, Osp24 functions as a cytoplasmic effector by competing against TaFROG for binding with TaSnRK1α, demonstrating the counteracting roles of orphan proteins of both host and fungal pathogens during their interactions.

Fusarium graminearum is the main causal agent of Fusarium head blight (FHB) on wheat and barley. In a previous study, a GIN4-like protein kinase gene, GIL1 , was found to be important for plant infection and sexual reproduction. In this study we further characterized the functions of GIL1 kinase in different developmental processes. The Δ gil1 mutants were reduced in growth, conidiation, and virulence, and formed whitish and compact colonies. Although phialide formation was rarely observed in the mutants, deletion of GIL1 resulted in increased hyphal branching and increased tolerance to cell wall and cell membrane stresses. The Δ gil1 mutants produced straight, elongated conidia lacking of distinct foot cells and being delayed in germination. Compared with the wild type, some compartments in the vegetative hyphae of Δ gil1 mutants had longer septal distances and increased number of nuclei, suggesting GIL1 is related to cytokinesis and septation. Localization of the GIL1-GFP fusion proteins to the septum and hyphal branching and fusion sites further supported its roles in septation and branching. Overall, our results indicate that GIL1 plays a role in vegetative growth and plant infection in F. graminearum , and is involved in septation and hyphal branching.

Fusarium graminearum is an important pathogen of wheat and barley. In addition to severe yield losses, infested grains are often contaminated with harmful mycotoxins. In this study, we characterized the functions of FgSSN3 kinase gene in different developmental and infection processes and gene regulation in F. graminearum. The FgSSN3 deletion mutant had a nutrient-dependent growth defects and abnormal conidium morphology. It was significantly reduced in DON production, TRI gene expression, and virulence. Deletion of FgSSN3 also resulted in up-regulation of HTF1 and PCS1 expression in juvenile cultures, and repression of TRI genes in DON-producing cultures. In addition, Fgssn3 was female sterile and defective in hypopodium formation and infectious growth. RNA-seq analysis showed that FgSsn3 is involved in the transcriptional regulation of a wide variety genes acting as either a repressor or activator. FgSsn3 physically interacted with C-type cyclin Cid1 and the cid1 mutant had similar phenotypes with Fgssn3, indicating that FgSsn3 and Cid1 form the CDK-cyclin pair as a component of the mediator complex in F. graminearum. Taken together, our results indicate that FgSSN3 is important for secondary metabolism, sexual reproduction, and plant infection, as a subunit of mediator complex contributing to transcriptional regulation of diverse genes.

Members of Cdc14 phosphatases are common in animals and fungi, but absent in plants. Although its orthologs are conserved in plant pathogenic fungi, their functions during infection are not clear. In this study, we showed that the CDC14 ortholog is important for pathogenesis and morphogenesis in Fusarium graminearum. FgCDC14 is required for normal cell division and septum formation and FgCdc14 possesses phosphatase activity with specificity for a subset of Cdk-type phosphorylation sites. The Fgcdc14 mutant was reduced in growth, conidiation, and ascospore formation. It was defective in ascosporogenesis and pathogenesis. Septation in Fgcdc14 was reduced and hyphal compartments contained multiple nuclei, indicating defects in the coordination between nuclear division and cytokinesis. Interestingly, foot cells of mutant conidia often differentiated into conidiogenous cells, resulting in the production of inter-connected conidia. In the interphase, FgCdc14-GFP localized to the nucleus and spindle-pole-body. Taken together, our results indicate that Cdc14 phosphatase functions in cell division and septum formation in F. graminearum, likely by counteracting Cdk phosphorylation, and is required for plant infection., (© 2015 John Wiley & Sons Ltd.)

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.

Fusarium head blight caused by Fusarium graminearum is one of the most destructive diseases of wheat and barley. Deoxynivalenol (DON) produced by the pathogen is an important mycotoxins and virulence factor. Because oxidative burst is a common defense response and reactive oxygen species (ROS) induces DON production, in this study, we characterized functional relationships of three stress-related transcription factor genes FgAP1, FgATF1 and FgSKN7. Although all of them played a role in tolerance to oxidative stress, deletion of FgAP1 or FgATF1 had no significant effect on DON production. In contrast, Fgskn7 mutants were reduced in DON production and defective in H2 O2 -induced TRI gene expression. The Fgap1 mutant had no detectable phenotype other than increased sensitivity to H2 O2 and Fgap1 Fgatf1 and Fgap1 Fgskn7 mutants lacked additional or more severe phenotypes than the single mutants. The Fgatf1, but not Fgskn7, mutant was significantly reduced in virulence and delayed in ascospore release. The Fgskn7 Fgatf1 double mutant had more severe defects in growth, conidiation and virulence than the Fgatf1 or Fgskn7 mutant. Instead of producing four-celled ascospores, it formed eight small, single-celled ascospores in each ascus. Therefore, FgSKN7 and FgATF1 must have overlapping functions in intracellular ROS signalling for growth, development and pathogenesis in F. graminearum., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The Kin1/Par-1/MARK kinases regulate various cellular processes in eukaryotic organisms. Kin1 orthologs are well conserved in fungal pathogens but none of them have been functionally characterized. Here, we show that KIN1 is important for pathogenesis and growth in two phytopathogenic fungi and that FgKin1 regulates ascospore germination and the localization of Tub1 β-tubulins in Fusarium graminearum. The Fgkin1 mutant and putative FgKIN1(S172A) kinase dead (nonactivatable) transformants were characterized for defects in plant infection, sexual and asexual reproduction, and stress responses. The localization of FgKin1 and two β-tubulins were examined in the wild-type and mutant backgrounds. Deletion of FgKIN1 resulted in reduced virulence and defects in ascospore germination and release. FgKin1 localized to the center of septal pores. FgKIN1 deletion had no effect on Tub2 microtubules but disrupted Tub1 localization. In the mutant, Tub1 appeared to be enriched in the nucleolus. In Magnaporthe oryzae, MoKin1 has similar functions in growth and infection and it also localizes to septal pores. The S172A mutation had no effect on the localization and function of FgKIN1 during sexual reproduction. These results indicate that FgKIN1 has kinase-dependent and independent functions and it specifically regulates Tub1 β-tubulins. FgKin1 plays a critical role in ascospore discharge, germination, and plant infection., (© 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.)

Arginine methylation of non-histone proteins by protein arginine methyltransferase (PRMT) has been shown to be important for various biological processes from yeast to human. Although PRMT genes are well conserved in fungi, none of them have been functionally characterized in plant pathogenic ascomycetes. In this study, we identified and characterized all of the four predicted PRMT genes in Fusarium graminearum, the causal agent of Fusarium head blight of wheat and barley. Whereas deletion of the other three PRMT genes had no obvious phenotypes, the Δamt1 mutant had pleiotropic defects. AMT1 is a predicted type I PRMT gene that is orthologous to HMT1 in Saccharomyces cerevisiae. The Δamt1 mutant was slightly reduced in vegetative growth but normal in asexual and sexual reproduction. It had increased sensitivities to oxidative and membrane stresses. DON mycotoxin production and virulence on flowering wheat heads also were reduced in the Δamt1 mutant. The introduction of the wild-type AMT1 allele fully complemented the defects of the Δamt1 mutant and Amt1-GFP fusion proteins mainly localized to the nucleus. Hrp1 and Nab2 are two hnRNPs in yeast that are methylated by Hmt1 for nuclear export. In F. graminearum, AMT1 is required for the nuclear export of FgHrp1 but not FgNab2, indicating that yeast and F. graminearum differ in the methylation and nucleo-cytoplasmic transport of hnRNP components. Because AMT2 also is a predicted type I PRMT with limited homology to yeast HMT1, we generated the Δamt1 Δamt2 double mutants. The Δamt1 single and Δamt1 Δamt2 double mutants had similar defects in all the phenotypes assayed, including reduced vegetative growth and virulence. Overall, data from this systematic analysis of PRMT genes suggest that AMT1, like its ortholog in yeast, is the predominant PRMT gene in F. graminearum and plays a role in hyphal growth, stress responses, and plant infection.

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.

As in other eukaryotes, protein kinases play major regulatory roles in filamentous fungi. Although the genomes of many plant pathogenic fungi have been sequenced, systematic characterization of their kinomes has not been reported. The wheat scab fungus Fusarium graminearum has 116 protein kinases (PK) genes. Although twenty of them appeared to be essential, we generated deletion mutants for the other 96 PK genes, including 12 orthologs of essential genes in yeast. All of the PK mutants were assayed for changes in 17 phenotypes, including growth, conidiation, pathogenesis, stress responses, and sexual reproduction. Overall, deletion of 64 PK genes resulted in at least one of the phenotypes examined, including three mutants blocked in conidiation and five mutants with increased tolerance to hyperosmotic stress. In total, 42 PK mutants were significantly reduced in virulence or non-pathogenic, including mutants deleted of key components of the cAMP signaling and three MAPK pathways. A number of these PK genes, including Fg03146 and Fg04770 that are unique to filamentous fungi, are dispensable for hyphal growth and likely encode novel fungal virulence factors. Ascospores play a critical role in the initiation of wheat scab. Twenty-six PK mutants were blocked in perithecia formation or aborted in ascosporogenesis. Additional 19 mutants were defective in ascospore release or morphology. Interestingly, F. graminearum contains two aurora kinase genes with distinct functions, which has not been reported in fungi. In addition, we used the interlog approach to predict the PK-PK and PK-protein interaction networks of F. graminearum. Several predicted interactions were verified with yeast two-hybrid or co-immunoprecipitation assays. To our knowledge, this is the first functional characterization of the kinome in plant pathogenic fungi. Protein kinase genes important for various aspects of growth, developmental, and infection processes in F. graminearum were identified in this study.

Members of Cdc14 phosphatases are common in animals and fungi, but absent in plants. Although its orthologs are conserved in plant pathogenic fungi, their functions during infection are not clear. In this study, we showed that the CDC 14 ortholog is important for pathogenesis and morphogenesis in F usarium graminearum. FgCDC 14 is required for normal cell division and septum formation and FgCdc14 possesses phosphatase activity with specificity for a subset of Cdk-type phosphorylation sites. The F gcdc14 mutant was reduced in growth, conidiation, and ascospore formation. It was defective in ascosporogenesis and pathogenesis. Septation in F gcdc14 was reduced and hyphal compartments contained multiple nuclei, indicating defects in the coordination between nuclear division and cytokinesis. Interestingly, foot cells of mutant conidia often differentiated into conidiogenous cells, resulting in the production of inter-connected conidia. In the interphase, FgCdc14- GFP localized to the nucleus and spindle-pole-body. Taken together, our results indicate that Cdc14 phosphatase functions in cell division and septum formation in F . graminearum, likely by counteracting Cdk phosphorylation, and is required for plant infection. [ABSTRACT FROM AUTHOR]

Fusarium head blight caused by F usarium graminearum is one of the most destructive diseases of wheat and barley. Deoxynivalenol ( DON) produced by the pathogen is an important mycotoxins and virulence factor. Because oxidative burst is a common defense response and reactive oxygen species ( ROS) induces DON production, in this study, we characterized functional relationships of three stress-related transcription factor genes FgAP 1, FgATF 1 and FgSKN 7. Although all of them played a role in tolerance to oxidative stress, deletion of FgAP 1 or FgATF 1 had no significant effect on DON production. In contrast, F gskn7 mutants were reduced in DON production and defective in H2O2-induced TRI gene expression. The F gap1 mutant had no detectable phenotype other than increased sensitivity to H2O2 and F gap1 F gatf1 and F gap1 F gskn7 mutants lacked additional or more severe phenotypes than the single mutants. The F gatf1, but not F gskn7, mutant was significantly reduced in virulence and delayed in ascospore release. The F gskn7 F gatf1 double mutant had more severe defects in growth, conidiation and virulence than the F gatf1 or F gskn7 mutant. Instead of producing four-celled ascospores, it formed eight small, single-celled ascospores in each ascus. Therefore, FgSKN 7 and FgATF 1 must have overlapping functions in intracellular ROS signalling for growth, development and pathogenesis in F . graminearum. [ABSTRACT FROM AUTHOR]

The Kin1/Par-1/ MARK kinases regulate various cellular processes in eukaryotic organisms. Kin1 orthologs are well conserved in fungal pathogens but none of them have been functionally characterized. Here, we show that KIN1 is important for pathogenesis and growth in two phytopathogenic fungi and that FgKin1 regulates ascospore germination and the localization of Tub1 β-tubulins in Fusarium graminearum., The Fgkin1 mutant and putative Fg KIN1S172A kinase dead (nonactivatable) transformants were characterized for defects in plant infection, sexual and asexual reproduction, and stress responses. The localization of FgKin1 and two β-tubulins were examined in the wild-type and mutant backgrounds., Deletion of Fg KIN1 resulted in reduced virulence and defects in ascospore germination and release. Fg Kin1 localized to the center of septal pores. Fg KIN1 deletion had no effect on Tub2 microtubules but disrupted Tub1 localization. In the mutant, Tub1 appeared to be enriched in the nucleolus. In Magnaporthe oryzae, MoKin1 has similar functions in growth and infection and it also localizes to septal pores. The S172A mutation had no effect on the localization and function of Fg KIN1 during sexual reproduction., These results indicate that Fg KIN1 has kinase-dependent and independent functions and it specifically regulates Tub1 β-tubulins. FgKin1 plays a critical role in ascospore discharge, germination, and plant infection. [ABSTRACT FROM AUTHOR]