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

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.

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

Genetics ◽

10.1093/genetics/160.1.169 ◽

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.

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Phenotype of a Mechanosensitive Channel Mutant, mid-1, in a Filamentous Fungus, Neurospora crassa

Eukaryotic Cell ◽

10.1128/ec.00411-07 ◽

2008 ◽

Vol 7 (4) ◽

pp. 647-655 ◽

Cited By ~ 32

Author(s):

Roger R. Lew ◽

Zohaib Abbas ◽

Marinela I. Anderca ◽

Stephen J. Free

Keyword(s):

Ion Transport ◽

Neurospora Crassa ◽

Carbon Sources ◽

Homologous Gene ◽

Ionophore A23187 ◽

Wild Type ◽

Knockout Mutant ◽

Lower Growth ◽

Filamentous Ascomycete ◽

Energy Dependent

ABSTRACT In the yeast Saccharomyces cerevisiae, the MID1 (mating-induced death) gene encodes a stretch-activated channel which is required for successful mating; the mutant phenotype is rescued by elevated extracellular calcium. Homologs of the MID1 gene are found in fungi that are morphologically complex compared to yeast, both Basidiomycetes and Ascomycetes. We explored the phenotype of a mid-1 knockout mutant in the filamentous ascomycete Neurospora crassa. The mutant exhibits lower growth vigor than the wild type (which is not rescued by replete calcium) and mates successfully. Thus, the role of the MID-1 protein differs from that of the homologous gene product in yeast. Hyphal cytology, growth on diverse carbon sources, turgor regulation, and circadian rhythms of the mid-1 mutant are all similar to those of the wild type. However, basal turgor is lower than wild type, as is the activity of the plasma membrane H+-ATPase (measured by cyanide [CN−]-induced depolarization of the energy-dependent component of the membrane potential). In addition, the mutant is unable to grow at low extracellular Ca2+ levels or when cytoplasmic Ca2+ is elevated with the Ca2+ ionophore A23187. We conclude that the MID-1 protein plays a role in regulation of ion transport via Ca2+ homeostasis and signaling. In the absence of normal ion transport activity, the mutant exhibits poorer growth.

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A network of MAP-Kinase pathways and transcription factors regulates cell-to-cell communication and cell wall integrity in Neurospora crassa

10.1101/281931 ◽

2018 ◽

Author(s):

Monika S. Fischer ◽

Vincent W. Wu ◽

Ji E. Lee ◽

Ronan C. O’Malley ◽

N. Louise Glass

Keyword(s):

Cell Wall ◽

Transcription Factors ◽

Map Kinase ◽

Neurospora Crassa ◽

Cell Fusion ◽

Regulatory Network ◽

Cell Communication ◽

Cell Wall Integrity ◽

Cell Integrity ◽

Map Kinase Pathways

ABSTRACTMaintenance of cell integrity and cell-to-cell communication are fundamental biological processes. Filamentous fungi, such as Neurospora crassa, depend on communication to locate compatible cells, coordinate cell fusion, and establish a robust hyphal network. Two MAP-Kinase pathways are essential for communication and cell fusion in N. crassa; the Cell Wall Integrity/MAK-1 pathway and the MAK-2 (signal response) pathway. Previous studies have demonstrated several points of cross talk between the MAK-1 and MAK-2 pathways, which is likely necessary for oordinating chemotropic growth toward an extracellular signal, and then mediating cell fusion. Canonical MAP-Kinase pathways begin with signal reception and end with a transcriptional response. Two transcription factors, ADV-1 and PP-1, are essential for communication and cell fusion. PP-1 is the conserved target of MAK-2, while it is unclear what targets ADV-1. We did RNAseq on Δadv-1, Δpp-1, and wild-type cells and found that ADV-1 and PP-1 have a shared regulon including many genes required for communication, cell fusion, growth, development, and stress response. We identified ADV-1 and PP-1 binding sites across the genome by adapting the in vitro method of DNA-Affinity Purification sequencing (DAP-seq) for N. crassa. To elucidate the regulatory network, we misexpressed each transcription factor in each upstream MAPK deletion mutant. Misexpression of adv-1 was sufficient to fully suppress the phenotype of the Δpp-1 mutant and partially suppress the phenotype of the Δmak-1 mutant. Collectively, our data demonstrate that the MAK-1-ADV-1 and MAK-2- PP-1 pathways form a tight regulatory network that maintains cell integrity and mediates communication and cell fusion.

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SO, a Protein Involved in Hyphal Fusion in Neurospora crassa, Localizes to Septal Plugs

Eukaryotic Cell ◽

10.1128/ec.00268-06 ◽

2006 ◽

Vol 6 (1) ◽

pp. 84-94 ◽

Cited By ~ 56

Author(s):

André Fleiβner ◽

N. Louise Glass

Keyword(s):

Cell Death ◽

Neurospora Crassa ◽

Colony Development ◽

Independent Manner ◽

Hyphal Fusion ◽

Filamentous Ascomycete ◽

Sealing Material ◽

Woronin Body ◽

Local Injury ◽

Extensive Damage

ABSTRACT The colony of a filamentous ascomycete fungus typically grows as a multinucleate syncytium. While this syncytial organization has developmental advantages, it bears the risk of extensive damage caused by local injury of hyphae. Loss of cytoplasm in injured hyphae is restricted by the fast and efficient sealing of the central pores of hyphal crosswalls, or septa, by a peroxisome-derived organelle called the Woronin body. The formation of septal plugs is also associated with development and leads to separation of certain parts of the colony. Septal plugs associated with developmental processes or aging hyphae typically occur by the accumulation of sealing material. Here we report that in Neurospora crassa, a protein necessary for hyphal fusion and proper colony development called SO (SOFT) localizes to septal plugs. In response to injury, SO accumulates at the septal plug in a Woronin body-independent manner. However, the presence of the Woronin body affects the speed of accumulation of SO at the septal pore. We determined that SO contributes to, but is not essential for, septal plugging. SO accumulation was also observed at septal plugs formed during hyphal aging and during programmed cell death mediated by genetic differences at heterokaryon incompatibility (het) loci.

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

mBio ◽

10.1128/mbio.00307-21 ◽

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.

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The ham-5, rcm-1 and rco-1 genes regulate hyphal fusion in Neurospora crassa

Microbiology ◽

10.1099/mic.0.040147-0 ◽

2010 ◽

Vol 156 (9) ◽

pp. 2621-2629 ◽

Cited By ~ 36

Author(s):

Mash'el Salman Aldabbous ◽

M. Gabriela Roca ◽

Angela Stout ◽

I-Ching Huang ◽

Nick D. Read ◽

...

Keyword(s):

Neurospora Crassa ◽

Protein Interactions ◽

Sexual Development ◽

Deletion Mutants ◽

Protein Protein Interactions ◽

Hyphal Fusion ◽

Acid Protein ◽

Hyphal Morphology ◽

Reduced Rate ◽

Amino Acid Protein

Mutants of Neurospora crassa unable to participate in vegetative hyphal fusion (anastomosis) were isolated and characterized. From this analysis, three genes, rcm-1, rco-1 and ham-5, were identified and shown to be required for hyphal fusion. The rcm-1 and rco-1 genes are homologues of the Saccharomyces cerevisiae SSN6 and TUP1 genes, which encode a dimeric transcription factor in yeast. We demonstrate that in N. crassa the rcm-1 and rco-1 genes are required for hyphal fusion and normal hyphal morphology, and influence both asexual and sexual development. The ham-5 gene encodes a 1686 amino acid protein with two putative WD40 domains, which might participate in protein–protein interactions. ham-5 deletion mutants had a reduced rate of hyphal extension and altered hyphal morphology, and were unable to produce the conidial anastomosis tubes that are required for hyphal fusion during colony initiation.

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Programmed Cell Death in Neurospora crassa

New Journal of Science ◽

10.1155/2014/479015 ◽

2014 ◽

Vol 2014 ◽

pp. 1-7 ◽

Cited By ~ 3

Author(s):

A. Pedro Gonçalves ◽

Arnaldo Videira

Keyword(s):

Hydrogen Peroxide ◽

Cell Death ◽

Programmed Cell Death ◽

Neurospora Crassa ◽

Filamentous Fungus ◽

Mammalian Cells ◽

Model Organism ◽

Chemical Induction ◽

Hyphal Fusion ◽

Heterokaryon Incompatibility

Programmed cell death has been studied for decades in mammalian cells, but simpler organisms, including prokaryotes, plants, and fungi, also undergo regulated forms of cell death. We highlight the usefulness of the filamentous fungus Neurospora crassa as a model organism for the study of programmed cell death. In N. crassa, cell death can be triggered genetically due to hyphal fusion between individuals with different allelic specificities at het loci, in a process called “heterokaryon incompatibility.” Chemical induction of cell death can also be achieved upon exposure to death-inducing agents like staurosporine, phytosphingosine, or hydrogen peroxide. A summary of the recent advances made by our and other groups on the discovery of the mechanisms and mediators underlying the process of cell death in N. crassa is presented.

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Ultrastructure and Morphology of the Neurospora Crassa Cell Wall Mutants, Slime And Slime X, Using Tem and Sem

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100090257 ◽

1976 ◽

Vol 34 ◽

pp. 54-55

Author(s):

Karen S. Howard ◽

H. D. Braymer ◽

M. D. Socolofsky ◽

S. A. Milligan

Keyword(s):

Cell Wall ◽

Neurospora Crassa ◽

Wild Type ◽

Morphological Comparison ◽

Small Areas ◽

Solid Media ◽

Thin Sectioning ◽

X Cells ◽

Mutant Cells ◽

Isolated Cell

The recently isolated cell wall mutant slime X of Neurospora crassa was prepared for ultrastructural and morphological comparison with the cell wall mutant slime. The purpose of this article is to discuss the methods of preparation for TEM and SEM observations, as well as to make a preliminary comparison of the two mutants.TEM: Cells of the slime mutant were prepared for thin sectioning by the method of Bigger, et al. Slime X cells were prepared in the same manner with the following two exceptions: the cells were embedded in 3% agar prior to fixation and the buffered solutions contained 5% sucrose throughout the procedure.SEM: Two methods were used to prepare mutant and wild type Neurospora for the SEM. First, single colonies of mutant cells and small areas of wild type hyphae were cut from solid media and fixed with OSO4 vapors similar to the procedure used by Harris, et al. with one alteration. The cell-containing agar blocks were dehydrated by immersion in 2,2-dimethoxypropane (DMP).

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