The NDR kinase DBF-2 is involved in regulation of mitosis, conidial development, and glycogen metabolism in Neurospora crassa.
ABSTRACT: Neurospora crassa dbf-2 encodes an NDR (nuclear Dbf2-related) protein kinase, homologous to LATS1, a core component of the Hippo pathway. This pathway plays important roles in restraining cell proliferation and promoting apoptosis in differentiating cells. Here, we demonstrate that DBF-2 is involved in three fundamental processes in a filamentous fungus: cell cycle regulation, glycogen biosynthesis, and conidiation. DBF-2 is predominantly localized to the nucleus, and most (approximately 60%) dbf-2 null mutant nuclei are delayed in mitosis, indicating that DBF-2 activity is required for properly completing the cell cycle. The dbf-2 mutant exhibits reduced basal hyphal extension rates accompanied by a carbon/nitrogen ratio-dependent bursting of hyphal tips, vast glycogen leakage, defects in aerial hypha formation, and impairment of all three asexual conidiation pathways in N. crassa. Our findings also indicate that DBF-2 is essential for sexual reproduction in a filamentous fungus. Defects in other Hippo and glycogen metabolism pathway components (mob-1, ccr-4, mst-1, and gsk-3) share similar phenotypes such as mitotic delay and decreased CDC-2 (cell division cycle 2) protein levels, massive hyphal swellings, hyphal tip bursting, glycogen leakage, and impaired conidiation. We propose that DBF-2 functions as a link between Hippo and glycogen metabolism pathways.
Project description:Proper cell division is essential for growth and development of uni- and multicellular organisms. The fungal septation initiation network (SIN) functions as kinase cascade that connects cell cycle progression with the initiation of cytokinesis. Miss-regulation of the homologous Hippo pathway in animals results in excessive cell proliferation and formation of tumors, underscoring the conservation of both pathways. How SIN proteins interact and transmit signals through the cascade is only beginning to be understood. Moreover, our understanding of septum formation and its regulation in filamentous fungi, which represent the vast majority of the fungal kingdom, is highly fragmentary. We determined that a tripartite kinase cascade, consisting of CDC-7, SID-1 and DBF-2, together with their regulatory subunits CDC-14 and MOB-1, is important for septum formation in the model mold Neurospora crassa. DBF-2 activity and septum formation requires auto-phosphorylation at Ser499 within the activation segment and phosphorylation of Thr671 in the hydrophobic motif by SID-1. Moreover, SID-1-stimulated DBF-2 activity is further enhanced by CDC-7, supporting a stepwise activation mechanism of the tripartite SIN kinase cascade in fungi. However, in contrast to the situation described for unicellular yeasts, the localization of the entire SIN cascade to spindle pole bodies is constitutive and cell cycle independent. Moreover, all SIN proteins except CDC-7 form cortical rings prior to septum initiation and localize to constricting septa. Thus, SIN localization and activity regulation significantly differs in unicellular versus syncytial ascomycete fungi.
Project description:In spite of its prevalence in animals and plants, endogenous nitric oxide (NO) has been rarely reported in fungi. We present here our observations on production of intracellular NO and its possible roles during development of Neurospora crassa, a model filamentous fungus. Intracellular NO was detected in hypha 8-16?hours after incubation in Vogel's minimal liquid media and conidiophores during conidiation using a fluorescent indicator (DAF-FM diacetate). Treatment with cPTIO, an NO scavenger, significantly reduced fluorescence levels and hindered hyphal growth in liquid media and conidiation, whereas exogenous NO enhanced hyphal extension on VM agar media and conidia formation. NO scavenging also dramatically diminished transcription of con-10 and con-13, genes preferentially expressed during conidiation. Our results suggest that intracellular NO is generated in young hypha growing in submerged culture and during conidia development and regulate mycelial development and conidia formation.
Project description:Mitogen-activated protein (MAP) kinase signaling pathways are ubiquitous and evolutionarily conserved in eukaryotic organisms. MAP kinase pathways are composed of a MAP kinase, a MAP kinase kinase, and a MAP kinase kinase kinase; activation is regulated by sequential phosphorylation. Components of three MAP kinase pathways have been identified by genome sequence analysis in the filamentous fungus Neurospora crassa. One of the predicted MAP kinases in N. crassa, MAK-2, shows similarity to Fus3p and Kss1p of Saccharomyces cerevisiae, which are involved in sexual reproduction and filamentation, respectively. In this study, we show that an N. crassa mutant disrupted in mak-2 exhibits a pleiotropic phenotype: derepressed conidiation, shortened aerial hyphae, lack of vegetative hyphal fusion, female sterility, and autonomous ascospore lethality. We assessed the phosphorylation of MAK-2 during conidial germination and early colony development. Peak levels of MAK-2 phosphorylation were most closely associated with germ tube elongation, branching, and hyphal fusion events between conidial germlings. A MAP kinase kinase kinase (NRC-1) is the predicted product of N. crassa nrc-1 locus and is a homologue of STE11 in S. cerevisiae. An nrc-1 mutant shares many of the same phenotypic traits as the mak-2 mutant and, in particular, is a hyphal fusion mutant. We show that MAK-2 phosphorylation during early colony development is dependent upon the presence of NRC-1 and postulate that phosphorylation of MAK-2 is required for hyphal fusion events that occur during conidial germination.
Project description:Highlights • Genome resequencing of classical genetic mutant strains associated gene with phenotype.• Neurospora crassa gene scumbo encodes a ceramide C9-methyltransferase.• Deletion of Neurospora crassa ceramide C9-methyltransferase encoding gene is viable. Using a legacy of genetic mutants of Neurospora crassa, paired with resequencing efforts through JGI, we have identified the gene responsible for the ‘scumbo’ mutant. This early morphological mutant was described as “Irregular flat, spreading growth with knobby protrusions and abnormal conidiation, but no free conidia. Mycelium usually appears yellowish rather than orange. Female fertile.” (Perkins, Radford et al. 2000). Our further investigation has found new insights into the identity and associated functions of scumbo as a ceramide C9 methyltransferase, previously annotated as “similar to cyclopropane-fatty-acyl-phospholipidsynthase”, encoded by the gene NCU07859. This enzyme performs a fungal-specific methyl modification of glycosyl-ceramides and has implications for membrane homeostasis and hyphal polarity in filamentous fungi. Graphical abstract Graphical abstract Image, graphical abstract.
Project description:Heterokaryon incompatibility (HI) is a nonself recognition phenomenon occurring in filamentous fungi that is important for limiting resource plundering and restricting viral transfer between strains. Nonself recognition and HI occurs during hyphal fusion between strains that differ at het loci. If two strains undergo hyphal fusion, but differ in allelic specificity at a het locus, the fusion cell is compartmentalized and undergoes a rapid programmed cell death (PCD). Incompatible heterokaryons show a macroscopic phenotype of slow growth and diminished conidiation, and a microscopic phenotype of hyphal compartmentation and cell death. To understand processes associated with HI and PCD, we used whole-genome microarrays for Neurospora crassa to assess transcriptional differences associated with induction of HI mediated by differences in het-c pin-c haplotype. Our data show that HI is a dynamic and transcriptionally active process. The production of reactive oxygen species is implicated in the execution of HI and PCD in N. crassa, as are several genes involved in phosphatidylinositol and calcium signalling pathways. However, genes encoding mammalian homologues of caspases or apoptosis-inducing factor (AIF) are not required for HI or programmed cell death. These data indicate that PCD during HI occurs via a novel and possibly fungal-specific mechanism, making this pathway an attractive drug target for control of fungal infections.
Project description:A non-self-recognition system called vegetative incompatibility is ubiquitous in filamentous fungi and is genetically regulated by het loci. Different fungal individuals are unable to form viable heterokaryons if they differ in allelic specificity at a het locus. To identify components of vegetative incompatibility mediated by allelic differences at the het-c locus of Neurospora crassa, we isolated mutants that suppressed phenotypic aspects of het-c vegetative incompatibility. Three deletion mutants were identified; the deletions overlapped each other in an ORF named vib-1 (vegetative incompatibility blocked). Mutations in vib-1 fully relieved growth inhibition and repression of conidiation conferred by het-c vegetative incompatibility and significantly reduced hyphal compartmentation and death rates. The vib-1 mutants displayed a profuse conidiation pattern, suggesting that VIB-1 is a regulator of conidiation. VIB-1 shares a region of similarity to PHOG, a possible phosphate nonrepressible acid phosphatase in Aspergillus nidulans. Native gel analysis of wild-type strains and vib-1 mutants indicated that vib-1 is not the structural gene for nonrepressible acid phosphatase, but rather may regulate nonrepressible acid phosphatase activity.
Project description:The life cycle of filamentous fungi generally comprises hyphal growth and asexual reproduction. Both growth and propagation processes are critical for invasion growth, spore dissemination, and virulence in fungal pathogens and for the production of secondary metabolites or for biomass accumulation in industrial filamentous fungi. The <u>C</u>CAAT-<u>b</u>inding <u>c</u>omplex (CBC) is a heterotrimeric transcription factor comprising three subunits, HapB, HapC, and HapE, and is highly conserved in fungi. Previous studies revealed that CBC regulates sterol metabolism by repressing several genes in the ergosterol biosynthetic pathway in the human fungal pathogen Aspergillus fumigatus. In the present study, we found dysfunction of CBC caused the abnormal asexual reproduction (conidiation) in submerged liquid culture. CBC suppresses the activation of the <i>brlA</i> gene in the central regulatory pathway for conidiation combined with its upstream regulators <i>fluG</i>, <i>flbD</i>, and <i>flbC</i> by binding to the 5'-CCAAT-3' motif within conidiation gene promoters, and lack of CBC member HapB results in the upregulation of these genes. Furthermore, when the expression of <i>brlA</i> or <i>flbC</i> is repressed, the submerged conidiation does not happen in the <i>hapB</i> mutant. Interestingly, deletion of HapB leads to enhanced transient cytosolic Ca<sup>2+</sup> levels and activates conidiation-positive inducer Ca<sup>2+</sup>-CrzA modules to enhance submerged conidiation, demonstrating that CrzA works with CBC as a reverse regulator of fungal conidiation. To the best of our knowledge, the finding of this study is the first report for the molecular switch mechanism between vegetative hyphal growth and asexual development regulated by CBC, in concert with Ca<sup>2+</sup>-CrzA signaling in A. fumigatus. <b>IMPORTANCE</b> A precisely timed switch between vegetative hyphal growth and asexual development is a crucial process for the filamentous fungal long-term survival, dissemination, biomass production, and virulence. However, under the submerged culture condition, filamentous fungi would undergo constant vegetative growth whereas asexual conidiation rarely occurs. Knowledge about possible regulators is scarce, and how they could inhibit conidiation in liquid culture is poorly understood. Here, we demonstrated that the transcription factor heterotrimeric CBC dominantly maintains vegetative growth in liquid-submerged cultures by directly suppressing the conidiation-inductive signal. In contrast, calcium and the transcription factor CrzA, are positive inducers of conidiation. Our new insights into the CBC and Ca<sup>2+</sup>-CrzA regulatory system for transition control in the submerged conidiation of A. fumigatus may have broad repercussions for all filamentous fungi. Moreover, our elucidation of the molecular mechanism for submerged conidiation may support new strategies to precisely control vegetative growth and asexual conidiation in aspergilli used in industry.
Project description:Transcription factors play an important role in fungal environmental adaptive process by promoting adjustment to challenging stimuli via gene modulation and activation of signaling networks. The transcription factor encoded by the pac-3/rim101/pacC gene is involved in pH regulation and is associated with a wide variety of cellular functions. The deletion of pac-3 affects fungal development. In Neurospora crassa, the ?pac-3 strain presents diminished aerial growth and reduced conidiation. However, the PAC-3-regulated genes associated with this altered phenotype have not been elucidated. In this study, we used RNA-seq to analyze the phenotypic plasticity induced after pac-3 deletion in the filamentous fungus N. crassa cultivated in media supplemented with sufficient or limited inorganic phosphate. Genes related to morphology, hyphal development, and conidiation were of particular interest in this study. Our results suggest a pac-3 dependency in gene regulation in a Pi-dependent manner. Furthermore, our analysis suggested that the fungus attempts to overcome the deletion effects in a ?pac-3 mutant through a complex combined regulatory mechanism. Finally, the modulatory responses observed in the ?pac-3 strain, a double mutant generated based on the ?mus-52 mutant strain, is strain-specific, highlighting that the phenotypic impact may be attributed to pac-3 absence despite the combined mus-52 deletion.
Project description:Heterotrimeric G proteins are critical regulators of growth and asexual and sexual development in the filamentous fungus Neurospora crassa. Three Gα subunits (GNA-1, GNA-2, and GNA-3), one Gβ subunit (GNB-1), and one Gγ subunit (GNG-1) have been functionally characterized, but genetic epistasis relationships between Gβ and Gα subunit genes have not been determined. Physical association between GNB-1 and FLAG-tagged GNG-1 has been previously demonstrated by coimmunoprecipitation, but knowledge of the Gα binding partners for the Gβγ dimer is currently lacking. In this study, the three N. crassa Gα subunits are analyzed for genetic epistasis with gnb-1 and for physical interaction with the Gβγ dimer. We created double mutants lacking one Gα gene and gnb-1 and introduced constitutively active, GTPase-deficient alleles for each Gα gene into the Δgnb-1 background. Genetic analysis revealed that gna-3 is epistatic to gnb-1 with regard to negative control of submerged conidiation. gnb-1 is epistatic to gna-2 and gna-3 for aerial hyphal height, while gnb-1 appears to act upstream of gna-1 and gna-2 during aerial conidiation. None of the activated Gα alleles restored female fertility to Δgnb-1 mutants, and the gna-3(Q208L) allele inhibited formation of female reproductive structures, consistent with a need for Gα proteins to cycle through the inactive GDP-bound form for these processes. Coimmunoprecipitation experiments using extracts from the gng-1-FLAG strain demonstrated that the three Gα proteins interact with the Gβγ dimer. The finding that the Gβγ dimer interacts with all three Gα proteins is supported by epistasis between gnb-1 and gna-1, gna-2, and gna-3 for at least one function.
Project description:The Aspergillus nidulans flbD gene encodes a protein with a Myb-like DNA-binding domain that is proposed to act in concert with other developmental regulators to control initiation of conidiophore development. We have identified a Neurospora crassa gene called rca-1 (regulator of conidiation in Aspergillus) based on its sequence similarity to flbD. We found that N. crassa rca-1 can complement the conidiation defect of an A. nidulans flbD mutant and that induced expression of rca-1 caused conidiation in submerged A. nidulans cultures just as was previously observed for overexpression of flbD. Thus, the N. crassa gene appears to be a functional homologue of A. nidulans flbD and this is the first demonstration of functional complementation of an A. nidulans sporulation defect using a gene from an evolutionarily distant fungus. However, deletion of the rca-1 gene in N. crassa had no major effect on growth rate, macroconidiation, microconidiation, or ascospore formation. The only phenotype displayed by the rca-1 mutant was straight or counterclockwise hyphal growth rather than the clockwise spiral growth observed for wild type. Thus, if rca-1 is involved in N. crassa development, its role is subtle or redundant.