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 response regulator BcSkn7 is required for vegetative differentiation and adaptation to oxidative and osmotic stresses in Botrytis cinerea

The high‐osmolarity glycerol pathway plays an important role in the responses of fungi to various environmental stresses. Saccharomyces cerevisiae Skn7 is a response regulator in the high‐osmolarity glycerol pathway, which regulates the oxidative stress response, cell cycle and cell wall biosynthesis. In this study, we characterized an Skn7 orthologue BcSkn7 in Botrytis cinerea. BcSKN7 can partly restore the growth defects of S. cerevisiae SKN7 mutant and vice versa. The BcSKN7 mutant (ΔBcSkn7‐1) revealed increased sensitivity to ionic osmotic and oxidative stresses and to ergosterol biosynthesis inhibitors. In addition, ΔBcSkn7‐1 was also impaired dramatically in conidiation and sclerotial formation. Western blot analysis showed that BcSkn7 positively regulated the phosphorylation of BcSak1 (the orthologue of S. cerevisiae Hog1) under osmotic stress, indicating that BcSkn7 is associated with the high‐osmolarity glycerol pathway in B. cinerea. In contrast with BcSak1, BcSkn7 is not involved in the regulation of B. cinerea virulence. All of the phenotypic defects of ΔBcSkn7‐1 are restored by genetic complementation of the mutant with the wild‐type BcSKN7. The results of this study indicate that BcSkn7 plays an important role in the regulation of vegetative differentiation and in the response to various stresses in B. cinerea.     

Shared and distinct functions of two Gti1/Pac2 family proteins in growth,morphogenesis and pathogenicity of Magnaporthe oryzae

Gti1/Pac2 are conserved family proteins that regulate morphogenic transition in yeasts such as Schizosaccharomyces pombe and Candida albicans, and they also control toxin production and pathogenicity in filamentous fungus Fusarium graminearum. To test the functions of Gti1/Pac2 paralogues MoGti1 and MoPac2 in the rice blast fungus Magnaporthe oryzae, we generated respective ΔMogti1 and ΔMopac2 mutant strains. We found that MoGti1 and MoPac2 exhibit shared and distinct roles in hyphal growth, conidiation, sexual reproduction, stress responses, surface hydrophobility, invasive hyphal growth and pathogenicity. Consistent with the putative conserved function of MoGti1, we showed that MoGti1‐GFP is localized to the nucleus, whereas MoPac2‐GFP is mainly found in the cytoplasm. In addition, we provided evidence that the nuclear localization of MoGti1 could be subject to regulation by MoPmk1 mitogen‐activated protein kinase. Moreover, we found that the reduced pathogenicity in the ΔMopac2 mutant corresponds with an increased expression of plant defence genes, including PR1a, AOS2, LOX1, PAD4, and CHT1. Taken together, our studies provide a comprehensive analysis of two similar but distinct Gti1/Pac2 family proteins in M. oryzae, which underlines the important yet conserved functions of these family proteins in plant pathogenic fungi.     

A two‐component histidine kinase Shk1 controls stress response,sclerotial formation and fungicide resistance in Sclerotinia sclerotiorum

Fungal histidine kinases (HKs) are involved in osmotic and oxidative stress responses, hyphal development, fungicide sensitivity and virulence. Members of HK class III are known to signal through the high‐osmolarity glycerol mitogen‐activated protein kinase (HOG MAPK). In this study, we characterized the Shk1 gene (SS1G_12694.3), which encodes a putative class III HK, from the plant pathogen Sclerotinia sclerotiorum. Disruption of Shk1 resulted in resistance to phenylpyrrole and dicarboximide fungicides and increased sensitivity to hyperosmotic stress and H₂O₂‐induced oxidative stress. The Shk1 mutant showed a significant reduction in vegetative hyphal growth and was unable to produce sclerotia. Quantitative real‐time polymerase chain reaction (qRT‐PCR and glycerol determination assays showed that the expression of SsHOG1 (the last kinase of the Hog pathway) and glycerol accumulation were regulated by the Shk1 gene, but PAK (p21‐activated kinase) was not. In addition, the Shk1 mutant showed no change in virulence. All the defects were restored by genetic complementation of the Shk1 deletion mutant with the wild‐type Shk1 gene. These findings indicate that Shk1 is involved in vegetative differentiation, sclerotial formation, glycerol accumulation and adaption to hyperosmotic and oxidative stresses, and to fungicides, in S. sclerotiorum. Taken together, our results demonstrate, for the first time, the role of two‐component HKs in Sclerotinia.     

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.     

FgCDC14 regulates cytokinesis,morphogenesis, and pathogenesis in Fusarium graminearum

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.     

The PKR regulatory subunit of protein kinase A (PKA) is involved in the regulation of growth,sexual and asexual development,and pathogenesis in Fusarium graminearum

Fusarium graminearum is a causal agent of wheat scab disease and a producer of deoxynivalenol (DON) mycotoxins. Treatment with exogenous cyclic adenosine monophosphate (cAMP) increases its DON production. In this study, to better understand the role of the cAMP–protein kinase A (PKA) pathway in F. graminearum, we functionally characterized the PKR gene encoding the regulatory subunit of PKA. Mutants deleted of PKR were viable, but showed severe defects in growth, conidiation and plant infection. The pkr mutant produced compact colonies with shorter aerial hyphae with an increased number of nuclei in hyphal compartments. Mutant conidia were morphologically abnormal and appeared to undergo rapid autophagy‐related cell death. The pkr mutant showed blocked perithecium development, but increased DON production. It had a disease index of less than unity and failed to spread to neighbouring spikelets. The mutant was unstable and spontaneous suppressors with a faster growth rate were often produced on older cultures. A total of 67 suppressor strains that grew faster than the original mutant were isolated. Three showed a similar growth rate and colony morphology to the wild‐type, but were still defective in conidiation. Sequencing analysis with 18 candidate PKA‐related genes in three representative suppressor strains identified mutations only in the CPK1 catalytic subunit gene. Further characterization showed that 10 of the other 64 suppressor strains also had mutations in CPK1. Overall, these results showed that PKR is important for the regulation of hyphal growth, reproduction, pathogenesis and DON production, and mutations in CPK1 are partially suppressive to the deletion of PKR in F. graminearum.     

The Colletotrichum graminicola striatin orthologue Str1 is necessary for anastomosis and is a virulence factor

Striatin family proteins are key regulators in signalling pathways in fungi and animals. These scaffold proteins contain four conserved domains: a caveolin‐binding domain, a coiled‐coil motif and a calmodulin‐binding domain at the N‐terminus, and a WD‐repeat domain at the C‐terminus. Fungal striatin orthologues are associated with sexual development, hyphal growth and plant pathogenesis. In Fusarium verticillioides, the striatin orthologue Fsr1 promotes virulence in the maize stalk. The relationship between fungal striatins and pathogenicity remains largely unexplored. In this study, we demonstrate that the Colletotrichum graminicola striatin orthologue Str1 is required for full stalk rot and leaf blight virulence in maize. Pathogenicity assays show that the striatin mutant strain (Δstr1) produces functional appressoria, but infection and colonization are attenuated. Additional phenotypes of the Δstr1 mutant include reduced radial growth and compromised hyphal fusion. In comparison with the wild‐type, Δstr1 also shows a defect in sexual development and produces fewer and shorter conidia. Together with the fact that F. verticillioides fsr1 can complement Δstr1, our results indicate that C. graminicola Str1 shares five phenotypes with striatin orthologues in other fungal species: hyphal growth, hyphal fusion, conidiation, sexual development and virulence. We propose that fungal striatins, like mammalian striatins, act as scaffolding molecules that cross‐link multiple signal transduction pathways.     

Sch9 is a major target of TORC1 in Saccharomyces cerevisiae

The Target of Rapamycin (TOR) protein is a Ser/Thr kinase that functions in two distinct multiprotein complexes: TORC1 and TORC2. These conserved complexes regulate many different aspects of cell growth in response to intracellular and extracellular cues. Here we report that the AGC kinase Sch9 is a substrate of yeast TORC1. Six amino acids in the C terminus of Sch9 are directly phosphorylated by TORC1. Phosphorylation of these residues is lost upon rapamycin treatment as well as carbon or nitrogen starvation and transiently reduced following application of osmotic, oxidative, or thermal stress. TORC1-dependent phosphorylation is required for Sch9 activity, and replacement of residues phosphorylated by TORC1 with Asp/Glu renders Sch9 activity TORC1 independent. Sch9 is required for TORC1 to properly regulate ribosome biogenesis, translation initiation, and entry into G0 phase, but not expression of Gln3-dependent genes. Our results suggest that Sch9 functions analogously to the mammalian TORC1 substrate S6K1 rather than the mTORC2 substrate PKB/Akt.     

Tor1, Sch9 and PKA downregulation in quiescence rely on Mtl1 to preserve mitochondrial integrity and cell survival

Here we show that Mtl1, member of the cell wall integrity pathway of Saccharomyces cerevisiae, plays a positive role in chronological life span (CLS). The absence of Mtl1 shortens CLS and causes impairment in the mitochondrial function. This is reflected in a descent in oxygen consumption during the postdiauxic state, an increase in the uncoupled respiration and mitochondrial membrane potential and also a descent in aconitase activity. We demonstrate that all these effects are a consequence of signalling defects suppressed by TOR1 (target of rapamycin) and SCH9 deletion and less efficiently by Protein kinase A (PKA) inactivation. Mtl1 also plays a role in the regulation of both Bcy1 stability and phosphorylation, mainly in response to glucose depletion. In postdiauxic phase and in conditions of glucose depletion, Mtl1 negatively regulates TOR1 function leading to Sch9 inactivation and Bcy1 phosphorylation converging in PKA inhibition. Slt2/Mpk1 kinase partially contributes to Bcy1 phosphorylation, although additional targets are not excluded. Mtl1 links mitochondrial dysfunction with TOR and PKA pathways in quiescence, glucose being the main signalling molecule.     

Ubiquitin regulates TORC1 in yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae the TOR complex 1 (TORC1) controls many growth‐related cellular processes and is essential for cell growth and proliferation. Macrolide antibiotic rapamycin, in complex with a cytosol protein named FKBP12, specifically inhibits TORC1, causing growth arrest. The FKBP12‐rapamycin complex interferes with TORC1 function by binding to the FRB domain of the TOR proteins. In an attempt to understand the role of the FRB domain in TOR function, we identified a single point mutation (Tor2^W2041R) in the FRB domain of Tor2 that renders yeast cells rapamycin resistant and temperature sensitive. At the permissive temperature, the Tor2 mutant protein is partially defective for binding with Kog1 and TORC1 is impaired for membrane association. At the restrictive temperature, Kog1 but not the Tor2 mutant protein, is rapidly degraded. Overexpression of ubiquitin stabilizes Kog1 and suppresses the growth defect associated with the tor2 mutant at the nonpremissive temperature. We find that ubiquitin binds non‐covalently to Kog1, prevents Kog1 from degradation and stabilizes TORC1. Our data reveal a unique role for ubiquitin in regulation of TORC1 and suggest that Kog1 requires association with the Tor proteins for stabilization.