scholarly journals The ham-2 Locus, Encoding a Putative Transmembrane Protein, Is Required for Hyphal Fusion in Neurospora crassa

Genetics ◽  
2002 ◽  
Vol 160 (1) ◽  
pp. 169-180
Author(s):  
Qijun Xiang ◽  
Carolyn Rasmussen ◽  
N Louise Glass
Keyword(s):  
Neurospora Crassa ◽  
Somatic Cell ◽  
Molecular Mechanism ◽  
Cell Fusion ◽  
Deletion Mutant ◽  
Transmembrane Protein ◽  
Wild Type ◽  
Vegetative Incompatibility ◽  
Hyphal Fusion ◽  
Aerial Hyphae

Abstract Somatic cell fusion is common during organogenesis in multicellular eukaryotes, although the molecular mechanism of cell fusion is poorly understood. In filamentous fungi, somatic cell fusion occurs during vegetative growth. Filamentous fungi grow as multinucleate hyphal tubes that undergo frequent hyphal fusion (anastomosis) during colony expansion, resulting in the formation of a hyphal network. The molecular mechanism of the hyphal fusion process and the role of networked hyphae in the growth and development of these organisms are unexplored questions. We use the filamentous fungus Neurospora crassa as a model to study the molecular mechanism of hyphal fusion. In this study, we identified a deletion mutant that was restricted in its ability to undergo both self-hyphal fusion and fusion with a different individual to form a heterokaryon. This deletion mutant displayed pleiotropic defects, including shortened aerial hyphae, altered conidiation pattern, female sterility, slow growth rate, lack of hyphal fusion, and suppression of vegetative incompatibility. Complementation with a single open reading frame (ORF) within the deletion region in this mutant restored near wild-type growth rates, female fertility, aerial hyphae formation, and hyphal fusion, but not vegetative incompatibility and wild-type conidiation pattern. This ORF, which we named ham-2 (for hyphal anastomosis), encodes a putative transmembrane protein that is highly conserved, but of unknown function among eukaryotes.

scholarly journals The so Locus Is Required for Vegetative Cell Fusion and Postfertilization Events in Neurospora crassa

2005 ◽  
Vol 4 (5) ◽  
pp. 920-930 ◽  
Author(s):  
André Fleißner ◽  
Sovan Sarkar ◽  
David J. Jacobson ◽  
M. Gabriela Roca ◽  
Nick D. Read ◽  
...  
Keyword(s):  
Neurospora Crassa ◽  
Cell Fusion ◽  
Protein Interactions ◽  
Cell Communication ◽  
Model Organism ◽  
Female Sterility ◽  
Wild Type ◽  
Hyphal Fusion ◽  
Filamentous Ascomycete ◽  
Ascomycete Yeast

ABSTRACT The process of cell fusion is a basic developmental feature found in most eukaryotic organisms. In filamentous fungi, cell fusion events play an important role during both vegetative growth and sexual reproduction. We employ the model organism Neurospora crassa to dissect the mechanisms of cell fusion and cell-cell communication involved in fusion processes. In this study, we characterized a mutant with a mutation in the gene so, which exhibits defects in cell fusion. The so mutant has a pleiotropic phenotype, including shortened aerial hyphae, an altered conidiation pattern, and female sterility. Using light microscopy and heterokaryon tests, the so mutant was shown to possess defects in germling and hyphal fusion. Although so produces conidial anastomosis tubes, so germlings did not home toward wild-type germlings nor were wild-type germlings attracted to so germlings. We employed a trichogyne attraction and fusion assay to determine whether the female sterility of the so mutant is caused by impaired communication or fusion failure between mating partners. so showed no defects in attraction or fusion between mating partners, indicating that so is specific for vegetative hyphal fusion and/or associated communication events. The so gene encodes a protein of unknown function, but which contains a WW domain; WW domains are predicted to be involved in protein-protein interactions. Database searches showed that so was conserved in the genomes of filamentous ascomycete fungi but was absent in ascomycete yeast and basidiomycete species.

scholarly journals The Predicted Mannosyltransferase GT69-2 Antagonizes RFW-1 To Regulate Cell Fusion in Neurospora crassa

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Yang Li ◽  
Jens Heller ◽  
A. Pedro Gonçalves ◽  
N. Louise Glass
Keyword(s):  
Cell Wall ◽  
Neurospora Crassa ◽  
Somatic Cell ◽  
Filamentous Fungi ◽  
Cell Fusion ◽  
Wild Type ◽  
Content Type ◽  
Asexual Sporulation ◽  
Somatic Cell Fusion ◽  
Cell Wall Remodeling

ABSTRACT Filamentous fungi undergo somatic cell fusion to create a syncytial, interconnected hyphal network which confers a fitness benefit during colony establishment. However, barriers to somatic cell fusion between genetically different cells have evolved that reduce invasion by parasites or exploitation by maladapted genetic entities (cheaters). Here, we identified a predicted mannosyltransferase, glycosyltransferase family 69 protein (GT69-2) that was required for somatic cell fusion in Neurospora crassa. Cells lacking GT69-2 prematurely ceased chemotropic signaling and failed to complete cell wall dissolution and membrane merger in pairings with wild-type cells or between Δgt69-2 cells (self fusion). However, loss-of-function mutations in the linked regulator of cell fusion and cell wall remodeling-1 (rfw-1) locus suppressed the self-cell-fusion defects of Δgt69-2 cells, although Δgt69-2 Δrfw-1 double mutants still failed to undergo fusion with wild-type cells. Both GT69-2 and RFW-1 localized to the Golgi apparatus. Genetic analyses indicated that RFW-1 negatively regulates cell wall remodeling-dependent processes, including cell wall dissolution during cell fusion, separation of conidia during asexual sporulation, and conidial germination. GT69-2 acts as an antagonizer to relieve or prevent negative functions on cell fusion by RFW-1. In Neurospora species and N. crassa populations, alleles of gt69-2 were highly polymorphic and fell into two discrete haplogroups. In all isolates within haplogroup I, rfw-1 was conserved and linked to gt69-2. All isolates within haplogroup II lacked rfw-1. These data indicated that gt69-2/rfw-1 are under balancing selection and provide new mechanisms regulating cell wall remodeling during cell fusion and conidial separation. IMPORTANCE Cell wall remodeling is a dynamic process that balances cell wall integrity versus cell wall dissolution. In filamentous fungi, cell wall dissolution is required for somatic cell fusion and conidial separation during asexual sporulation. In the filamentous fungus Neurospora crassa, allorecognition checkpoints regulate the cell fusion process between genetically different cells. Our study revealed two linked loci with transspecies polymorphisms and under coevolution, rfw-1 and gt69-2, which form a coordinated system to regulate cell wall remodeling during somatic cell fusion, conidial separation, and asexual spore germination. RFW-1 acts as a negative regulator of these three processes, while GT69-2 functions antagonistically to RFW-1. Our findings provide new insight into the mechanisms involved in regulation of fungal cell wall remodeling during growth and development.

scholarly journals N-3-Hydroxy Dodecanoyl-DL-homoserine Lactone (OH-dDHL) Triggers Apoptosis of Bone Marrow-Derived Macrophages through the ER- and Mitochondria-Mediated Pathways

2021 ◽  
Vol 22 (14) ◽  
pp. 7565
Author(s):  
Kyungho Woo ◽  
Dong Ho Kim ◽  
Man Hwan Oh ◽  
Ho Sung Park ◽  
Chul Hee Choi
Keyword(s):  
Bone Marrow ◽  
Quorum Sensing ◽  
Deletion Mutant ◽  
Transmembrane Protein ◽  
Cell Communication ◽  
Homoserine Lactone ◽  
Host Responses ◽  
Wild Type ◽  
Mutant Strains

Quorum sensing of Acinetobacter nosocomialis for cell-to-cell communication produces N-3-hydroxy dodecanoyl-DL-homoserine lactone (OH-dDHL) by an AnoR/I two-component system. However, OH-dDHL-driven apoptotic mechanisms in hosts have not been clearly defined. Here, we investigated the induction of apoptosis signaling pathways in bone marrow-derived macrophages treated with synthetic OH-dDHL. Moreover, the quorum-sensing system for virulence regulation was evaluated in vivo using wild-type and anoI-deletion mutant strains. OH-dDHL decreased the viability of macrophage and epithelial cells in dose- and time-dependent manners. OH-dDHL induced Ca2+ efflux and caspase-12 activation by ER stress transmembrane protein (IRE1 and ATF6a p50) aggregation and induced mitochondrial dysfunction through reactive oxygen species (ROS) production, which caused cytochrome c to leak. Pretreatment with a pan-caspase inhibitor reduced caspase-3, -8, and -9, which were activated by OH-dDHL. Pro-inflammatory cytokine and paraoxonase-2 (PON2) gene expression were increased by OH-dDHL. We showed that the anoI-deletion mutant strains have less intracellular invasion compared to the wild-type strain, and their virulence, such as colonization and dissemination, was decreased in vivo. Consequently, these findings revealed that OH-dDHL, as a virulence factor, contributes to bacterial infection and survival as well as the modification of host responses in the early stages of infection.

scholarly journals Mutational Activation of a Gαi Causes Uncontrolled Proliferation of Aerial Hyphae and Increased Sensitivity to Heat and Oxidative Stress in Neurospora crassa

Genetics ◽  
1999 ◽  
Vol 151 (1) ◽  
pp. 107-117
Author(s):  
Qi Yang ◽  
Katherine A Borkovich
Keyword(s):  
Signal Transduction ◽  
Neurospora Crassa ◽  
Signal Transduction Pathway ◽  
Sexual Cycle ◽  
Intracellular Camp ◽  
Wild Type ◽  
Independent Functions ◽  
Aerial Hyphae ◽  
Mutational Activation ◽  
Camp Levels

Abstract Heterotrimeric G proteins, consisting of α, β, and γ subunits, transduce environmental signals through coupling to plasma membrane-localized receptors. We previously reported that the filamentous fungus Neurospora crassa possesses a Gα protein, GNA-1, that is a member of the Gαi superfamily. Deletion of gna-1 leads to defects in apical extension, differentiation of asexual spores, sensitivity to hyperosmotic media, and female fertility. In addition, Δgna-1 strains have lower intracellular cAMP levels under conditions that promote morphological abnormalities. To further define the function of GNA-1 in signal transduction in N. crassa, we examined properties of strains with mutationally activated gna-1 alleles (R178C or Q204L) as the only source of GNA-1 protein. These mutations are predicted to inhibit the GTPase activity of GNA-1 and lead to constitutive signaling. In the sexual cycle, gna-1R178C and gna-1Q204L strains are female-fertile, but produce fewer and larger perithecia than wild type. During asexual development, gna-1R178C and gna-1Q204L strains elaborate abundant, long aerial hyphae, produce less conidia, and possess lower levels of carotenoid pigments in comparison to wild-type controls. Furthermore, gna-1R178C and gna-1Q204L strains are more sensitive to heat shock and exposure to hydrogen peroxide than wild-type strains, while Δgna-1 mutants are more resistant. In contrast to Δgna-1 mutants, gna-1R178C and gna-1Q204L strains have higher steady-state levels of cAMP than wild type. The results suggest that GNA-1 possesses several Gβγ-independent functions in N. crassa. We propose that GNA-1 mediates signal transduction pathway(s) that regulate aerial hyphae development and sensitivity to heat and oxidative stresses, possibly through modulation of cAMP levels.

scholarly journals Cell Biology of Conidial Anastomosis Tubes in Neurospora crassa

2005 ◽  
Vol 4 (5) ◽  
pp. 911-919 ◽  
Author(s):  
M. Gabriela Roca ◽  
Jochen Arlt ◽  
Chris E. Jeffree ◽  
Nick D. Read
Keyword(s):  
Neurospora Crassa ◽  
Optical Tweezers ◽  
Cell Biology ◽  
Transmembrane Protein ◽  
Mitogen Activated Protein Kinase ◽  
Cellular Element ◽  
Hyphal Fusion ◽  
Self Recognition ◽  
Mitogen Activated Protein ◽  
Germ Tubes

ABSTRACT Although hyphal fusion has been well documented in mature colonies of filamentous fungi, it has been little studied during colony establishment. Here we show that specialized hyphae, called conidial anastomosis tubes (CATs), are produced by all types of conidia and by conidial germ tubes of Neurospora crassa. The CAT is shown to be a cellular element that is morphologically and physiologically distinct from a germ tube and under separate genetic control. In contrast to germ tubes, CATs are thinner, shorter, lack branches, exhibit determinate growth, and home toward each other. Evidence for an extracellular CAT inducer derived from conidia was obtained because CAT formation was reduced at low conidial concentrations. A cr-1 mutant lacking cyclic AMP (cAMP) produced CATs, indicating that the inducer is not cAMP. Evidence that the transduction of the CAT inducer signal involves a putative transmembrane protein (HAM-2) and the MAK-2 and NRC-1 proteins of a mitogen-activated protein kinase signaling pathway was obtained because ham-2, mak-2, and nrc-1 mutants lacked CATs. Optical tweezers were used in a novel experimental assay to micromanipulate whole conidia and germlings to analyze chemoattraction between CATs during homing. Strains of the same and opposite mating type were shown to home toward each other. The cr-1 mutant also underwent normal homing, indicating that cAMP is not the chemoattractant. ham-2, mak-2, and nrc-1 macroconidia did not attract CATs of the wild type. Fusion between CATs of opposite mating types was partially inhibited, providing evidence of non-self-recognition prior to fusion. Microtubules and nuclei passed through fused CATs.

scholarly journals Characterization of Neurospora crassa cyclic AMP phosphodiesterase activated by calmodulin

1985 ◽  
Vol 232 (2) ◽  
pp. 425-430 ◽  
Author(s):  
M T Téllez-Iñón ◽  
R M Ulloa ◽  
G C Glikin ◽  
H N Torres
Keyword(s):  
Cyclic Amp ◽  
Neurospora Crassa ◽  
Cyclic Nucleotide ◽  
Neuroleptic Drugs ◽  
Wild Type ◽  
Cyclic Amp Phosphodiesterase ◽  
Aerial Hyphae

Activation of cyclic AMP phosphodiesterase I by brain or Neurospora calmodulin was studied. The stimulation required micromolar concentrations of Ca2+, and it was observed at cyclic AMP concentrations between 0.1 and 500 microM. Activation was blocked by EDTA and some neuroleptic drugs such as chlorpromazine and fluphenazine. These drugs inhibit the elongation of N. crassa wild-type aerial hyphae. These results reinforce the evidence towards the recognition of Ca2+-calmodulin as one of the systems controlling cyclic nucleotide concentrations in Neurospora.

scholarly journals Identification and Characterization of LFD-2, a Predicted Fringe Protein Required for Membrane Integrity during Cell Fusion in Neurospora crassa

2015 ◽  
Vol 14 (3) ◽  
pp. 265-277 ◽  
Author(s):  
Javier Palma-Guerrero ◽  
Jiuhai Zhao ◽  
A. Pedro Gonçalves ◽  
Trevor L. Starr ◽  
N. Louise Glass
Keyword(s):  
Neurospora Crassa ◽  
Somatic Cell ◽  
Cell Fusion ◽  
Cell Lysis ◽  
Extracellular Calcium ◽  
Data Set ◽  
Content Type ◽  
Calcium Dependent ◽  
Somatic Cell Fusion ◽  
Dependent Cell

ABSTRACTThe molecular mechanisms of membrane merger during somatic cell fusion in eukaryotic species are poorly understood. In the filamentous fungusNeurospora crassa, somatic cell fusion occurs between genetically identical germinated asexual spores (germlings) and between hyphae to form the interconnected network characteristic of a filamentous fungal colony. InN. crassa, two proteins have been identified to function at the step of membrane fusion during somatic cell fusion: PRM1 and LFD-1. The absence of either one of these two proteins results in an increase of germling pairs arrested during cell fusion with tightly appressed plasma membranes and an increase in the frequency of cell lysis of adhered germlings. The level of cell lysis in ΔPrm1or Δlfd-1germlings is dependent on the extracellular calcium concentration. An available transcriptional profile data set was used to identify genes encoding predicted transmembrane proteins that showed reduced expression levels in germlings cultured in the absence of extracellular calcium. From these analyses, we identified a mutant (lfd-2, forlatefusiondefect-2) that showed a calcium-dependent cell lysis phenotype.lfd-2encodes a protein with a Fringe domain and showed endoplasmic reticulum and Golgi membrane localization. The deletion of an additional gene predicted to encode a low-affinity calcium transporter,fig1, also resulted in a strain that showed a calcium-dependent cell lysis phenotype. Genetic analyses showed that LFD-2 and FIG1 likely function in separate pathways to regulate aspects of membrane merger and repair during cell fusion.

scholarly journals The Golgi-resident protease Kex2 acts in conjunction with Prm1 to facilitate cell fusion during yeast mating

2007 ◽  
Vol 176 (2) ◽  
pp. 209-222 ◽  
Author(s):  
Maxwell G. Heiman ◽  
Alex Engel ◽  
Peter Walter
Keyword(s):  
Cell Wall ◽  
Cell Fusion ◽  
Plasma Membranes ◽  
Transmembrane Protein ◽  
Wild Type ◽  
Substantial Fraction ◽  
Yeast Mating ◽  
Fusion Defect ◽  
Mediate Cell ◽  
Wall Components

The molecular machines that mediate cell fusion are unknown. Previously, we identified a multispanning transmembrane protein, Prm1 (pheromone-regulated membrane protein 1), that acts during yeast mating (Heiman, M.G., and P. Walter. 2000. J. Cell Biol. 151:719–730). Without Prm1, a substantial fraction of mating pairs arrest with their plasma membranes tightly apposed yet unfused. In this study, we show that lack of the Golgi-resident protease Kex2 strongly enhances the cell fusion defect of Prm1-deficient mating pairs and causes a mild fusion defect in otherwise wild-type mating pairs. Lack of the Kex1 protease but not the Ste13 protease results in similar defects. Δkex2 and Δkex1 fusion defects were suppressed by osmotic support, a trait shared with mutants defective in cell wall remodeling. In contrast, other cell wall mutants do not enhance the Δprm1 fusion defect. Electron microscopy of Δkex2-derived mating pairs revealed novel extracellular blebs at presumptive sites of fusion. Kex2 and Kex1 may promote cell fusion by proteolytically processing substrates that act in parallel to Prm1 as an alternative fusion machine, as cell wall components, or both.

scholarly journals STUDIES ON A CYTOPLASMIC CHARACTER IN NEUROSPORA CRASSA

1965 ◽  
Vol 26 (2) ◽  
pp. 413-425 ◽  
Author(s):  
Laura Garnjobst ◽  
J.F. Wilson ◽  
E. L. Tatum
Keyword(s):  
Growth Rate ◽  
Oxygen Consumption ◽  
Neurospora Crassa ◽  
Great Variability ◽  
Wild Type ◽  
Aerial Hyphae ◽  
Genetic Strains ◽  
First Time ◽  
Slow Growing ◽  
Microinjection Techniques

Two morphologically distinctive slow growing strains of Neurospora crassa have been isolated and studied. These, abn-1 and abn-2, differ from wild type in that their growth rates are greatly reduced and often irregular, aerial hyphae are absent, conidia are extremely rare, and no protoperithecia are formed. Growth was not improved by addition of any nutrients tested, oxygen consumption was similar to that of wild type, and cytochrome c appeared abnormally high, and b low or absent. Both abn strains gave rise only to normal progeny in crosses with normal strains. The abn characteristics appear in heterocaryons, and have been transmitted to other genetic strains by means of heterocaryosis followed by plating of conidia. Conidia formed by such heterocaryons typically showed low viability, and gave rise to cultures with great variability in growth rate, morphology, and survival. Even apparently normal derived cultures often later became abnormal or died. It is concluded that the abnormal characteristics are determined primarily by cytoplasmic factors. This conclusion was strengthened by the transmission of the typical characteristics to normal strains by microinjection of cytoplasm from abn cultures, even without demonstrable transfer of nuclei. This constitutes the first time microinjection techniques have been successfully applied to the analysis of a cytoplasmic character in Neurospora.

scholarly journals Shared and Independent Roles for a Gαi Protein and Adenylyl Cyclase in Regulating Development and Stress Responses in Neurospora crassa

2002 ◽  
Vol 1 (4) ◽  
pp. 634-642 ◽  
Author(s):  
F. Douglas Ivey ◽  
Ann M. Kays ◽  
Katherine A. Borkovich
Keyword(s):  
Neurospora Crassa ◽  
Adenylyl Cyclase ◽  
Stress Responses ◽  
Growth And Development ◽  
Submerged Culture ◽  
Wild Type Strain ◽  
Wild Type ◽  
Liquid Cultures ◽  
Aerial Hyphae ◽  
Gαi Protein

ABSTRACT Growth and development are regulated using cyclic AMP (cAMP)-dependent and -independent pathways in Neurospora crassa. The cr-1 adenylyl cyclase mutant lacks detectable cAMP and exhibits numerous defects, including colonial growth habit, short aerial hyphae, premature conidiation on plates, inappropriate conidiation in submerged culture, and increased thermotolerance. Evidence suggests that the heterotrimeric Gα protein GNA-1 is a direct positive regulator of adenylyl cyclase. Δgna-1 strains are female-sterile, and Δgna-1 strains have reduced apical extension rates on normal and hyperosmotic medium, greater resistance to oxidative and heat stress, and stunted aerial hyphae compared to the wild-type strain. In this study, a Δgna-1 cr-1 double mutant was analyzed to differentiate cAMP-dependent and -independent signaling pathways regulated by GNA-1. Δgna-1 cr-1 mutants have severely restricted colonial growth and do not produce aerial hyphae on plates or in standing liquid cultures. Addition of cAMP to plates or standing liquid cultures rescues cr-1, but not Δgna-1 cr-1, defects, which is consistent with previous results demonstrating that Δgna-1 mutants do not respond to exogenous cAMP. The females of all strains carrying the Δgna-1 mutation are sterile; however, unlike cr-1 and Δgna-1 strains, the Δgna-1 cr-1 mutant does not produce protoperithecia. The Δgna-1 and cr-1 mutations were synergistic with respect to inappropriate conidiation during growth in submerged culture. Thermotolerance followed the order wild type < Δgna-1 < cr-1 = Δgna-1 cr-1, consistent with a cAMP-dependent process. Taken together, the results suggest that in general, GNA-1 and CR-1 regulate N. crassa growth and development using parallel pathways, while thermotolerance is largely dependent on cAMP.

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